Antagonists targeting the tgf-b pathway

ABSTRACT

Antitumor antagonists that bind specifically to immune checkpoint regulators, angiogenesis pathway regulators and/or TGF pathway regulators are disclosed. Also disclosed are methods for treating proliferative disorders, infections, and immunological disorders with the antitumor antagonists described herein.

This application is a continuation application of Ser. No. 16/706,187,filed Dec. 6, 2019, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/869,111, filed on Jul. 1, 2019. The entirety ofall the aforementioned application is expressly incorporated herein byreference for all purposes.

FIELD

The present application relates generally to cancer treatment and, inparticular, to bispecific inhibitors capable of modulating pathwaysassociated with tumorigenesis, tumor immunity, and angiogenesis.

BACKGROUND

The inability of the host to eliminate cancer cells remains a majorproblem. Although an increasing number of therapeutic monoclonalantibodies have been approved for treatment of various cancers,emergence of resistance to these antibodies is frequently observed,given the many different molecular pathways underlying cancer growth andprogression to metastasis. Although the immune system is the principalmechanism of cancer prevention, cancer cells counteractimmunosurveillance. Natural control mechanisms have been identified thatlimit T-cell activation so as to prevent collateral damage resultingfrom unrestrained T-cell activity. This process has been exploited bytumor cells to evade immune responses. Restoring the capacity of immuneeffector cells, especially T cells, to recognize and eliminate cancer isa major objective in immunotherapy.

The need exists for improved therapeutic binding antagonists orantibodies and methods of treating cancer and chronic viral infectionswith such reagents.

SUMMARY

The inventors of the present application have discovered thatantagonists bearing a TGFβ1 RII ECD domain exhibit unacceptable levelsof proteolytic degradation or clipping over time. One aspect of thepresent application relates to antitumor antagonists containing a firsttargeting domain specifically binding TGFβ1 and a second targetingdomain specifically binding PD-1 or PD-L1, wherein the first targetingdomain contains a mutated TGF-β1 RII extracellular domain (mutated ECD)that significantly reduces proteolytic cleavage of the antitumorantagonist.

Another aspect of the present application relates to an antitumorantagonist containing a first targeting domain comprising a TGFβ pathwayinhibitor, and a second targeting domain that binds specifically toVEGF.

Another aspect of the present application relates to methods fortreating a cell proliferative disorder in a subject. The methodcomprises the step of administering to a subject in need of suchtreatment an effective amount the antitumor antagonist of the presentapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows complementary determining region (CDR) sequences of certainanti-PD-1 mabs. The corresponding framework region (FR) sequences arelisted in FIG. 53 as SEQ ID NOS: 176-205.

FIGS. 2A-2B show several embodiments of anti-PD-1 antibody variabledomain sequences.

FIG. 3 shows CDR sequences of certain anti-PD-L1 mabs. The correspondingFR sequences are listed in FIG. 53 as SEQ ID NOS: 206-228.

FIGS. 4A-4C show several embodiments of anti-PD-L1 antibody variabledomain sequences.

FIGS. 5A-5B show four different bispecific antitumor antagonistconfigurations, Bi-PB-1, Bi-PLB-1, Bi-PB-2 and Bi-PLB-2, each comprising(1) anti-PD-1 or anti-PD-L1 variable regions and (2) a TGF-β RII ECDdomain.

FIG. 6 shows exemplary functional domain sequences corresponding to thebispecific antibodies in FIG. 5.

FIG. 7 shows exemplary heavy chain (HC) and light chain (LC) sequencescorresponding to selected bispecific antibodies in FIG. 5.

FIGS. 8A-8C depict three different bispecific antagonists, Bi-AB-1,Bi-A1B-1 and Bi-ZB-1, respectively, each comprising a carboxy-terminalTGF-β1 RII extracellular domain (ECD) in a mutant IgG1 (K447A) scaffold.Bi-AB-1 and Bi-A1B-1 both contain amino-terminal anti-VEGF variableregions (VH1, VL1) from Avastin/bevacizumab; Bi-A1B-1 includes two aminoacid substitutions in the VH region (E6Q, L11V). Bi-ZB-1 contains anamino terminal aflibercept domain upstream of an IgG1 Fc (K447A) region.

FIGS. 9A and 9B show the various functional domain sequences present inthe bispecific antagonists depicted in FIG. 8.

FIG. 10 shows the heavy chain (HC) and light chain (LC) amino acidsequences corresponding to the bispecific antagonists depicted in FIG.8.

FIG. 11 summarizes the arrangement of functional domains in thebispecific antagonists depicted in FIG. 8.

FIG. 12 is a Coomassie blue stained polyacrylamide gel showing improvedexpression levels of bispecific antibody antagonists Bi-AB-1 andBi-A1B-1 containing E6Q and L11V mutations when transiently transfectedinto HEK293 cells as determined by non-reducing polyacrylamide gelelectrophoresis (PAGE).

FIG. 13A shows size exclusion chromatography (SEC) profiles of Bi-AB-1and Bi-A1B-1. FIG. 13B shows that protein A purified Bi-AB-1 andBi-A1B-1 have low levels of high molecular weight (HMW, 1%, 1%,respectively) and low molecular weight (LMW, 0.4%, 0.4%, respectively)species in comparison to dimers (98.6%, 98.7%, respectively)

FIGS. 14A and 14B show PAGE gels of transiently expressed Bi-ZB-1 undernon-reducing (FIG. 14A) and reducing (FIG. 14B) conditions

FIG. 15 shows a size exclusion chromatography (SEC) profile showing thatprotein A purified Bi-ZB-1 has low levels of high molecular weight (HMW,3.4%) and low molecular weight (LMW, 0.5%) species in comparison todimers (96.1%).

FIG. 16 shows the results of a cell-based assay in which recombinantHEK-293 cells expressing human VEGFR2 and firefly luciferase under thecontrol of NFAT response elements were stimulated with huVEGF165 in thepresence of serial dilutions of two anti-VEGF antagonists, Bi-A1B-1 andantibody A. Bioactivity was determined by measuring the decrease in theamount of luciferase-mediated luminescence.

FIG. 17 is an ELISA analysis demonstrating simultaneous binding TGF-β1and VEGF165 by Bi-A1B-1 in which huTGF-β1 coated 96 well platesincubated with serially diluted samples Bi-A1B-1, followed bybiotinylated huVEGF165, whereby bound molecules were detected byStreptavidin-HRP using a TMB substrate.

FIG. 18 shows the results of a cell-based assay in which recombinantHEK-293 cells expressing human TGF-β1 RII receptor and fireflyluciferase under the control of a SMAD response element and stimulatedwith huTGF-β1 in the presence of serial dilutions of Bi-A1B-1, Bi-ZB-1,and a control, each containing a TGF-β1 RII extracellular domain (ECD)fusion. Bioactivity was determined by a decrease in the amount ofluciferase-mediated luminescence.

FIG. 19 is a pharmacokinetic profile showing the in vivo half-life(T_(1/2)) of the bispecific antagonist Bi-A1B-1 following a tail veininjection into 6-10 week old female CD1 mice. The Bi-A1B-1 antagonist inserum was recovered at various times post-injection and subjected toanalysis by ELISA.

FIGS. 20A-20B depict two bispecific antitumor antagonists, Bi-PB-1.2(FIG. 20A) and Bi-PLB-1.2 (FIG. 20B), each comprising antibody backbone(IgG4 K447A or IgG1 K447A) with variable region domains from anti-PD-1and anti-PD-L1, respectively, and additionally including a TGF-β-RII ECDfused to the carboxy-terminal end of each heavy chain CH3 region.

FIG. 21 shows functional domain sequences present in the bispecificantibodies in FIGS. 20A and 20B.

FIG. 22 show the heavy chain (HC) and light chain (LC) amino acidsequences corresponding to the bispecific antagonists depicted in FIGS.20A and 20B.

FIG. 23 summarizes the arrangement of functional domains in thebispecific antagonists depicted in FIGS. 20A and 20B.

FIG. 24A shows a non-denaturing polyacrylamide gel (PAGE) analysisshowing the expression of Bi-PB-1.2 and Bi-PLB-1.2 in a transientexpression system in comparison to 1 μg of parental control antibody(2P17). FIG. 24B depicts size exclusion chromatography (SEC) profilesshowing of protein A purified Bi-PB-1.2, Bi-PLB-1.2 and ananti-PDL1-TGF-β1 RII ECD benchmark molecule. FIG. 24C shows thatBi-PB-1.2, Bi-PLB-1.2 and the anti-PDL1-TGF-β1 RII ECD benchmarkmolecule have low levels of high molecular weight (HMW) low molecularweight (LMW) species in comparison to dimers.

FIGS. 25A-25B show that the Dimer, HMW and LMW forms of Bi-PB-1.2 (FIG.25A) and Bi-PLB-1.2 (FIG. 25B) exhibit good stability for at least 4weeks at 4° C.

FIGS. 26A-26E show the results of PD-1 and TGF-β1 binding to Bi-PB-1.2(26A, 26B, respectively) and corresponding anti-PD-1 andanti-PDL1-TGF-β1 RII ECD benchmark molecules (26C, 26D, respectively),along with their resultant binding affinity constants (26E).

FIGS. 27A-27E show the results of PD-L1 and TGF-β1 binding to Bi-PLB-1.2(27A, 27B, respectively) and the anti-PDL1-TGF-β1 RII ECD benchmarkmolecule (27C, 27D), along with their resultant binding affinityconstants (27E).

FIG. 28A shows an ELISA assays demonstrating simultaneous binding ofTGF-β1 and PD-1 by Bi-PB-1.2 in which huTGF-β1 coated 96 well plateswere incubated with serially diluted samples of Bi-PB-1.2, followed bybiotinylated hu PD-1, whereby bound molecules were detected byStreptavidin-HRP using a TMB substrate. FIG. 28B shows an ELISA assaydemonstrating simultaneous binding of TGF-β1 and PD-L1 by Bi-PLB-1.2 inwhich huTGF-β1 coated 96 well plates were incubated with seriallydiluted samples of Bi-PLB-1.2, followed by biotinylated hu PD-L1,whereby bound molecules were detected by Streptavidin-HRP using a TMBsubstrate.

FIG. 29A shows the ability of Bi-PB-1.2 and anti-PD-1 benchmark antibodyto block binding of PD-1 to PD-L1. FIG. 29B shows the results of acell-based assay showing the ability of serial dilutions of Bi-PB-1.2and anti-PDL1-TGF-β1 RII ECD benchmark molecule to block the ability ofTGF-β1 to activate luciferase expression under the control of a SMADresponse element. Bioactivity was determined by a decrease in the amountof luciferase-mediated luminescence.

FIGS. 30A-30B show binding of Bi-PB-1.2 and both anti-human PD-1 andanti-cyno PD-1 benchmark antibodies to human PD-1 (FIG. 30A) andcynomolgus PD-1 (FIG. 30B), along with their corresponding EC50 valuesreflecting the half maximal effective concentrations (EC₅₀) producing aresponse halfway between the baseline and maximum response with respectto binding human PD-1 and cyno PD-1, respectively.

FIG. 31A shows the ability of Bi-PLB-1.2 and anti-PD-L1 benchmarkantibody to block binding of PD-L1 to PD-1. FIG. 31B shows the resultsof a cell-based assay showing the ability of serial dilutions ofBi-PLB-1.2 and anti-PDL1-TGF-β1 RII ECD benchmark molecule to block theability of TGF-β1 to activate luciferase expression under the control ofa SMAD response element. Bioactivity was determined by a decrease in theamount of luciferase-mediated luminescence.

FIGS. 32A-32B show binding of Bi-PLB-1.2 and both anti-human PD-L1 andanti-cyno PD-L1 benchmark antibodies to human PD-L1 (FIG. 32A) and cynoPD-L1 (FIG. 32B), along with their corresponding EC50 values reflectingthe half maximal effective concentrations (EC₅₀) producing a responsehalfway between the baseline and maximum response with respect tobinding human PD-L1 and cyno PD-L1, respectively.

FIGS. 33A-33B show increased IFN-γ secretion from human PBMCs (Donor 1,FIG. 33A; Donor 2, FIG. 33B) with Bi-PB-1.2 relative to the negativecontrol treatments. FIGS. 33C-33D show increased IL-2 section from humanPBMCs (Donor 1, FIG. 33C; Donor 2, FIG. 33D) with Bi-PB-1.2 relative tothe negative control treatments.

FIGS. 34A-34B show increased IFN-γ secretion from human PBMCs (Donor 8,FIG. 34A; Donor 9, FIG. 34B) with Bi-PLB-1.2 relative to the parentalanti-PD-L1 antibody and the negative control treatments. FIGS. 34C-34Dshow increased IL-2 section from human PBMCs (Donor 8, FIG. 34C; Donor9, FIG. 34D) with Bi-PLB-1.2 relative to the parental anti-PD-L1antibody and negative control treatments.

FIGS. 35A-35F show that Bi-PB-1.2 and Bi-PLB-1.2 exhibit improvedpharmacokinetic profiles relative to a benchmark antibody following atail vein injection into 6-10 week old female CD1 mice. The antibodyantagonists, Bi-PB-1.2, Bi-PLB-1.2, and Benchmark were recovered fromserum at various times post-injection and subjected to analysis by ELISA(FIGS. 35A, 35C, 35E, respectively) and Western blot (FIGS. 35B, 35D,35F, respectively).

FIG. 36 shows that the percentage of low molecular weight (LMW) speciesof Bi-PB-1.2 (PD1-TGFβ-ECD) produced by stably transfected CHO cellsincreases over time stored at 4° C., while the percentage of dimerspecies decreases over time, consistent with increased clipping over asdetermined by size-exclusion ultra-high performance liquidchromatography (SE-UHPLC).

FIG. 37 shows size exclusion chromatograph (SEC) profiles of thebispecific antagonist, Bi-PB-1.2 (PD1-TGF-β1 RII ECD) and a PLB-1benchmark (BM) antibody expressed in stably transformed CHO cells, whichdepict a shoulder in the main peak consistent with increased clippingcompared to Bi-PD-1.2 produced by HEK293 transiently transfected cells.

FIG. 38 shows SEC profiles of Bi-PB-1.2 fractions obtained bycation-exchange chromatography (CEX).

FIG. 39 identifies fragments and clipped fragments identified by massspectrometry showing that Bi-A1B-1 and Bi-PB-1.2 have similar clippingsites.

FIGS. 40A and 40B show the location of clip sites in the heavy chainamino acid sequences of Bi-A1B-1 and Bi-PB-1.2, respectively, based onmass spectrometry.

FIGS. 41A-41C shows various mutant TGF-β1 RII ECD sequences forinvestigating clipping of the wild-type TGF-β1 RII ECD.

FIGS. 42A-41H show Bi-PB-1.2 heavy chain sequences containing the mutantTGF-β1 RII ECD sequences in FIGS. 41A-41C. Analysis of these mutants ispresented in FIGS. 46-52 below.

FIG. 43 shows Bi-PLB-1.2 heavy chain sequences containing selectedmutantTGF-β1 RII ECD sequences in FIGS. 41A and 41C. Analysis of thesemutants is presented in FIG. 52 below.

FIGS. 44A-44D show Bi-A1B-1 heavy chain sequences containing selectedmutant TGF-β1 RII ECD sequences in FIGS. 49, 50 and 52 below.

FIG. 45 shows the amino acids targeted in the mutant TGF-β1 RII ECDsequences represented in FIGS. 41A-41C.

FIG. 46A-46C depict reducing polyacrylamide gel (PAGE) analyses showingsimilar expression levels and yields from the Bi-PB-1.2 TGFβ1 ECDmutants compared to the parental wild-type Bi-PB-1.2 following proteinA-affinity purification of molecules from stable Chinese hamster ovarycell (CHO) transformants.

FIG. 47A shows SEC profiles corresponding to Bi-PB-1.2 and the mutants,Bi-PB-1.2-Δ7, Bi-PB-1.2B to Bi-PB-1.2H, which show reduced clipping (noshoulder) of the mutants compared to Bi-PB-1.2. FIG. 47B shows SECprofiles corresponding to Bi-PB-1.2 and the mutants, Bi-PB-1.2-Δ15, andBi-PB-1.2-Δ20, which show reduced clipping (no shoulder) of the mutantscompared to Bi-PB-1.2.

FIG. 48 shows the percentages of high molecular weight (HMW) species,dimers, and low molecular weight (LMW) species reflected in the SECprofiles in FIGS. 47A-47B.

FIG. 49 shows the results of a cell-based assay showing the ability ofserial dilutions of the Bi-PB-1.2 and Bi-A1B-1 variants and controls(i.e., parental Bi-PB-1.2, Bi-A1B-1 and PLB-BM (Benchmark) and negativecontrol antibody) produced by HEK293 transient transfection to block theability of TGF-β1 to activate luciferase expression under the control ofa SMAD response element. Bioactivity was determined by a decrease in theamount of luciferase-mediated luminescence.

FIG. 50 shows the IC50s of the Bi-PB-1.2 and Bi-A1B-1 variants andparental controls reflecting their ability to block TGF-β1-mediatedactivation of luciferase expression under the control of a SMAD responseelement. Bioactivity was determined by a decrease in the amount ofluciferase-mediated luminescence.

FIGS. 51A-51C show that Bi-PB-1.2 (FIG. 51A), Bi-PB-1.2C (FIG. 51B) andBi-PB-1.2D (FIG. 51C) have similar pharmacokinetics in paired mice,characterized by a T of about 5-8 days.

FIG. 52 shows FIGS. 52A & 52B a reducing polyacrylamide gel (PAGE)analysis showing reduced levels of clipped fragments from the C variantscompared to the parental wild-type Bi-PB-1.2, Bi-PLB-1.2 and Bi-A1B-1.

FIG. 53 shows framework regions (FRs) corresponding to the anti-PD-1CDRs (SEQ ID NOs: 176-205) and anti-PD-L1 CDRs (SEQ ID NOs: 206-228) inFIGS. 1 and 3.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide” includes“one or more” peptides or a “plurality” of such peptides. With respectto the teachings in the present application, any issued patent or patentapplication publication described in this application is expresslyincorporated by reference herein.

As used herein, the term “PD-1” refers to any form of PD-1 and variantsthereof that retain at least part of the activity of PD-1. Unlessindicated differently, such as by specific reference to human PD-1, PD-1includes all mammalian species of native sequence PD-1, e.g., human,canine, feline, equine, and bovine.

As used herein, the term “PD-L1” refers to any form of PD-L1 andvariants thereof that retain at least part of the activity of PD-L1.Unless indicated differently, such as by specific reference to humanPD-L1, PD-L1 includes all mammalian species of native sequence PD-L1,e.g., human, canine, feline, equine, and bovine.

The term “agonist” refers to a substance which promotes (i.e., induces,causes, enhances, or increases) the biological activity or effect ofanother molecule. The term agonist encompasses substances which bindreceptor, such as antibody, and substances which promote receptorfunction without binding thereto (e.g., by activating an associatedprotein).

The term “antagonist” or “inhibitor” refers to a substance thatprevents, blocks, inhibits, neutralizes, or reduces a biologicalactivity or effect of another molecule, such as a receptor or ligand.

As used herein, the term “antibody” refers to a polypeptide or apolypeptide complex that specifically recognizes and binds to antigenthrough one or more immunoglobulin variable regions. Antibody can be awhole antibody, antigen binding fragment or a single chain thereof.

The term “antibody” encompasses various broad classes of polypeptidesthat can be distinguished biochemically. Those skilled in the art willappreciate that heavy chains are classified as alpha, delta, epsilon,gamma, and mu, or α, δ, ε, γ and μ) with some subclasses among them(e.g., γ1-γ4). It is the nature of this chain that determines the“class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, etc.are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are within the scope of thepresent disclosure, the following discussion will generally be directedto the IgG class of immunoglobulin molecules.

Antibodies or antibody antagonists of the present application mayinclude, but are not limited to polyclonal, monoclonal, multispecific,bispecific, trispecific, human, humanized, primatized, chimeric and,single chain antibodies. Antibodies disclosed herein may be from anyanimal origin, including birds and mammals. Preferably, the antibodiesare human, murine, rat, donkey, rabbit, goat, guinea pig, camel, llama,horse, or chicken antibodies. In some embodiments, the variable regionmay be condricthoid in origin (e.g., from sharks).

The terms “antibody fragment” or “antigen-binding fragment” are usedwith reference to a portion of antibody, such as F(ab′)2, F(ab)2, Fab′,Fab, Fv, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library andanti-idiotypic (anti-Id) antibodies. Regardless of structure, antibodyfragment binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” includes DARTs and diabodies. Theterm “antibody fragment” also includes any synthetic or geneticallyengineered proteins comprising immunoglobulin variable regions that actlike antibody by binding to a specific antigen to form a complex. A“single-chain fragment variable” or “scFv” refers to a fusion protein ofthe variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins. In some aspects, the regions are connected with a shortlinker peptide of ten to about 25 amino acids. The linker can be rich inglycine for flexibility, as well as serine or threonine for solubility,and can either connect the N-terminus of the VH with the C-terminus ofthe VL, or vice versa. This protein retains the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker. With regard to IgGs, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwhere the light chains bracket the heavy chains starting at the mouth ofthe “Y” and continuing through the variable region.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention, the numbering of the constant region domains in conventionalantibodies increases as they become more distal from the antigen-bindingsite or amino-terminus of the antibody. In conventional antibodies, theN-terminal portion is a variable region and at the C-terminal portion isa constant region; the CH3 and CL domains actually comprise thecarboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VL chains (i.e.HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3). In some instances, e.g.,certain immunoglobulin molecules are derived from camelid species orengineered based on camelid immunoglobulins. Alternatively, animmunoglobulin molecule may consist of heavy chains only with no lightchains or light chains only with no heavy chains.

In naturally occurring antibodies, the six CDRs present in eachantigen-binding domain are short, non-contiguous sequences of aminoacids that are specifically positioned to form the antigen-bindingdomain as the antibody assumes its three dimensional configuration in anaqueous environment. The remainder of the amino acids in theantigen-binding domains, referred to as “framework” regions, show lessinter-molecular variability. The framework regions largely adopt aβ-sheet conformation and the CDRs form loops which connect, and in somecases form part of, the β-sheet structure. Thus, framework regions actto form a scaffold that provides for positioning the CDRs in correctorientation by inter-chain, non-covalent interactions. Theantigen-binding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto its cognate epitope. The amino acids comprising the CDRs and theframework regions, respectively, can be readily identified for any givenheavy or light chain variable region by one of ordinary skill in theart, since they have been precisely defined.

As used herein, the terms “VH1” and “VH2” refer to immunoglobulin heavychain variable domains corresponding to two different bindingspecificities. Likewise, the terms “VL1” and “VL2” refer to light chainvariable domains corresponding to two different binding specificities.When used together, it is to be understood that VH1 and VL1 regionsdefine a common binding specificity and that VH2 and VL2 domains definea second binding specificity.

The term “framework region (FR)” as used herein refers to variabledomain residues other than the CDR residues. Each variable domaintypically has four FRs flanking the corresponding CDRs. For example, aVH domain typically has four HFRs: HFR, HFR2, HFR3 and HFR4 flanking thethree HCDRs in the configuration ofHFR-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. Similarly, an LH domain typicallyhas four LFRs flanking the three LCDRs in the configuration of:LFR-LCDR-LFR2-LCDR2-LFR3-LCDR3-LFR4. Exemplary FRs that may be utilizedin the antagonists described herein are summarized in FIG. 53.

Light chains are classified as either kappa or lambda (K, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “Fc” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

As used herein, the terms “light chain constant region” or “CL” are usedinterchangeably herein with reference to amino acid sequences derivedfrom an antibody light chain. Preferably, the light chain constantregion comprises at least one of a constant kappa domain or constantlambda domain.

As used herein, the term “heavy chain constant region” includes aminoacid sequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain constant region comprises at least one of: aCH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region)domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.For example, antigen-binding polypeptide for use in the disclosure maycomprise a polypeptide chain comprising a CH1 domain; a polypeptidechain comprising a CH1 domain, at least a portion of a hinge domain, anda CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH3 domain, or a polypeptide chain comprising aCH1 domain, at least a portion of a hinge domain, a CH2 domain, and aCH3 domain. In some embodiments, a polypeptide of the disclosurecomprises a polypeptide chain comprising a CH3 domain. Further, antibodyfor use in the disclosure may lack at least a portion of a CH2 domain(e.g., all or part of a CH2 domain). It should be understood that theheavy chain constant region may be modified such that they vary in aminoacid sequence from the naturally occurring immunoglobulin molecule.

For example, as reflected in the disclosure herein below, Applicant hasfound that the CH3 domain can tolerate or accommodate significantinsertions (e.g., greater than 100 aa) in the Fc loop of the CH3 domain(see e.g., Bi-PB-2, Bi-PLB-2 in FIG. 5B). Therefore, in the presentapplication, any of the disclosed inhibitor domains may be similarlyinserted in the Fc loop in a manner analogous to the insertion of theTGFβ1 RII ECD domain in the Fc loop as in the heavy chain sequences setforth in SEQ ID NOS: 151-154.

The heavy chain constant region of antibody disclosed herein may bederived from different immunoglobulin molecules. For example, a heavychain constant region of a polypeptide may comprise a CH1 domain derivedfrom an IgG1 molecule and a hinge region derived from an IgG3 molecule.In another example, a heavy chain constant region can comprise a hingeregion derived, in part, from an IgG1 molecule and, in part, from anIgG3 molecule. In another example, a heavy chain portion can comprise achimeric hinge derived, in part, from an IgG1 molecule and, in part,from an IgG4 molecule.

A “light chain-heavy chain pair” refers to the collection of a lightchain and heavy chain that can form a dimer through a disulfide bondbetween the CL domain of the light chain and the CH1 domain of the heavychain.

The subunit structures and three dimensional configurations of theconstant regions of the various immunoglobulin classes are well known.As used herein, the term “VH domain” includes the amino terminalvariable domain of an immunoglobulin heavy chain and the term “CH1domain” includes the first (most amino terminal) constant region domainof an immunoglobulin heavy chain. The CH1 domain is adjacent to the VHdomain and is amino terminal to the hinge region of an immunoglobulinheavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of antibody using conventional numbering schemes (residues 244 to 360,Kabat numbering system; and residues 231-340, EU numbering system). TheCH2 domain is unique in that it is not closely paired with anotherdomain. Rather, two N-linked branched carbohydrate chains are interposedbetween the two CH2 domains of an intact native IgG molecule. The CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains.

As used herein the term “disulfide bond” includes a covalent bond formedbetween two sulfur atoms. The amino acid cysteine comprises a thiolgroup that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, a “variant” of antibody, antibody fragment or antibodydomain refers to antibody, antibody fragment or antibody domain that (1)shares a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% with the original antibody, antibody fragment or antibody domain,and (2) binds specifically to the same target that the originalantibody, antibody fragment or antibody domain binds specifically. Itshould be understood that where a measure of sequence identity ispresented in the form of the phrase “at least x % identical” or “atleast x % identity”, such an embodiment includes any and all wholenumber percentages equal to or above the lower limit. Further it shouldbe understood that where an amino acid sequence is presented in thepresent application, it should be construed as additionally disclosingor embracing amino acid sequences having a sequence identity of at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity to that amino acid sequence.

It should be understood that where a sequence homology range ispresented herein, as in e.g., the phrase “about 80% to about 100%”, suchan embodiment includes any and all subranges within, wherein the lowernumber can be any whole number between 80 and 100.

As used herein, the phrase “humanized antibody” refers to antibodyderived from a non-human antibody, typically a mouse monoclonalantibody. Alternatively, a humanized antibody may be derived from achimeric antibody that retains or substantially retains the antigenbinding properties of the parental, non-human, antibody but whichexhibits diminished immunogenicity as compared to the parental antibodywhen administered to humans.

As used herein, the phrase “chimeric antibody,” refers to antibody wherethe immunoreactive region or site is obtained or derived from a firstspecies and the constant region (which may be intact, partial ormodified in accordance with the instant disclosure) is obtained from asecond species. In certain embodiments the target binding region or sitewill be from a non-human source (e.g., mouse or primate) and theconstant region is human.

Included within the scope of the multispecific antibodies of the presentapplication are various compositions and methodologies, includingasymmetric IgG-like antibodies (e.g., triomab/quadroma, TrionPharma/Fresenius Biotech); knobs-into-holes antibodies (Genentech);Cross MAbs (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y(Genentech); strand exchange engineered domain (SEED) body (EMD Serono;Biolonic, Merus); Fab-exchanged antibodies (Genmab), symmetric IgG-likeantibodies (e.g. dual targeting (DT)-Ig (GSK/Domantis); two-in-oneantibody (Genentech); crosslinked MAbs (Karmanos Cancer Center), mAb2(F-star); Cov X-body (Cov X/Pfizer); dual variable domain (DVD)-Igfusions (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab(Medimmune/AZ); BsAb (ZymoGenetics); HERCULES (Biogen Idec,TvAb, Roche);scFv/Fc fusions; SCORPION (Emergent BioSolutions/Trubion,ZymoGenetics/BMS); dual affinity retargeting technology (Fc-DART),MacroGenics; dual (scFv)2-Fabs (National Research Center for AntibodyMedicine); F(ab)2 fusions (Medarex/AMGEN); dual-action or Bis-Fab(Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech);scFv- and diabody-based antibodies (e.g., bispecific T cell engagers(BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DARTs(MacroGenics); single-chain diabodies; TCR-like antibodies (AIT,Receptor Logics); human serum albumin scFv fusion (Merrimack); COMBODIES(Epigen Biotech); and IgG/non-IgG fusions (e.g., immunocytokines(EMDSerono, Philogen, ImmunGene, ImmunoMedics).

By “specifically binds” or “has specificity to”, it is generally meantthat antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition,antibody is said to “specifically bind” to an epitope when it binds tothat epitope via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.” In some embodiments,antibody or antibody fragment “has specificity to” antigen if theantibody or antibody fragment forms a complex with the antigen with adissociation constant (Kd) of 10-6M or less, 10-7M or less, 10-8M orless, 10-9M or less, or 10-10M or less.

The phrase “immune checkpoint regulator” refers to a functional class ofagents, which inhibit or stimulate signaling through an immunecheckpoint. An “immune checkpoint regulator” includes cell surfacereceptors and their associated ligands, which together provide a meansfor inhibiting or stimulating signaling pathways associated with T-cellactivation. Immune checkpoint regulators that are cell surface receptorsare typically members of either the TNF receptor or B7 superfamiliesthat can induce checkpoint signaling pathways to suppress immuneresponses, including agents which bind to negative co-stimulatorymolecules including without limitation PD-1, TIGIT, LAG-3, TIM-3, BTLA,VISTA, CTLA-4, and their respective ligands.

The phrases “immune checkpoint regulator antagonist”, “immune checkpointbinding antagonist” and “immune checkpoint antagonist” are usedinterchangeably herein with reference to a class of agents thatinterfere with (or inhibit) the activity of an immune checkpointregulator so that, as a result of the binding to the checkpointregulator or its ligand, signaling through the checkpoint regulatorreceptor is blocked or inhibited. By inhibiting this signaling,immune-suppression can be reversed so that T cell immunity againstcancer cells can be re-established or enhanced. Exemplary immunecheckpoint antagonists include, but are not limited to PD-1 and itsligands, PD-L1 and PD-L2; TIGIT and its CD155 ligand, PVR; LAG-3 and itsligands, including liver sinusoidal endothelial cell lectin (LSECtin)and Galectin-3; CTLA-4 and its ligands, B7-1 and B7-2; TIM-3 and itsligand, Galectin-9; CD122 and its CD122R ligand; CD70, B7H3, B and Tlymphocyte attenuator (BTLA), and VISTA. Immune checkpoint regulatorantagonists may include antibody fragments, peptide inhibitors, dominantnegative peptides and small molecule drugs, either in isolated forms oras part of a fusion protein or conjugate.

The phrases “immune checkpoint binding agonist” and “immune checkpointagonist” are used interchangeably herein with reference to a class ofagents that stimulate the activity of an immune checkpoint regulator sothat, as a result of the binding to the checkpoint regulator or itsligand, signaling through the checkpoint regulator receptor isstimulated. By stimulating this signaling, T cell immunity againstcancer cells can be re-established or enhanced. Exemplary immunecheckpoint regulator agonists include, but are not limited to members ofthe tumor necrosis factor (TNF) receptor superfamily, such as CD27,CD40, OX40 (CD 134), glucocorticoid-induced TNFR family-related protein(GITR), and 4-1BB (CD137) and their ligands. Additional checkpointregulator agonists belong to the B7-CD28 superfamily, including CD28 andICOS. Immune checkpoint regulator may include antibody fragments,peptide inhibitors, dominant negative peptides and small molecule drugs,either in isolated forms or as part of a fusion protein or conjugate.

The term “antagonist antibody” refers to antibody that binds to a targetand prevents or reduces the biological effect of that target. In someembodiments, the term can denote antibody that prevents the target,e.g., PD-1, to which it is bound from performing a biological function.

As used herein, an “anti-PD-1 antagonist antibody” refers to antibodythat is able to inhibit PD-1 biological activity and/or downstreamevents(s) mediated by PD-1. Anti-PD-1 antagonist antibodies encompassantibodies that block, antagonize, suppress or reduce (to any degreeincluding significantly) PD-1 biological activity, including downstreamevents mediated by PD-1, such as PD-1 binding and downstream signaling,inhibition of T cell proliferation, inhibition of T cell activation,inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition ofTNF secretion, induction of IL-10, and inhibition of anti-tumor immuneresponses. For purposes of the present invention, it will be explicitlyunderstood that the term “anti-PD-1 antagonist antibody”(interchangeably termed “antagonist PD-1 antibody”, “antagonistanti-PD-1 antibody” or “PD-1 antagonist antibody”) encompasses all thepreviously identified terms, titles, and functional states andcharacteristics whereby PD-1 itself, a PD-1 biological activity, or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree. In some embodiments,anti-PD-1 antagonist antibody binds PD-1 and upregulates anti-tumorimmune response.

As used herein, an “anti-PD-L1 antagonist antibody” refers to antibodythat is able to inhibit PD-L1 biological activity and/or downstreamevents(s) mediated by PD-L1. Anti-PD-L1 antagonist antibodies encompassantibodies that block, antagonize, suppress or reduce (to any degreeincluding significantly) PD-L1 biological activity, including downstreamevents mediated by PD-L1, such as PD-L1 binding and downstreamsignaling, inhibition of T cell proliferation, inhibition of T cellactivation, inhibition of IFN secretion, inhibition of IL-2 secretion,inhibition of TNF secretion, induction of IL-10, and inhibition ofanti-tumor immune responses. For purposes of the present invention, itwill be explicitly understood that the term “anti-PD-L1 antagonistantibody” (interchangeably termed “antagonist PD-L1 antibody”,“antagonist anti-PD-L1 antibody” or “PD-L1 antagonist antibody”)encompasses all the previously identified terms, titles, and functionalstates and characteristics whereby PD-L1 itself, a PD-L1 biologicalactivity, or the consequences of the biological activity, aresubstantially nullified, decreased, or neutralized in any meaningfuldegree. The anti-PD-L1 antagonist is designed to bind PD-L1 andupregulate anti-tumor immune responses.

The phrases “dominant-negative protein” or “dominant-negative peptide”refer to a protein or peptide derived from a wild type protein that hasbeen genetically modified by mutation and/or deletion so that themodified protein or peptide interferes with the function of theendogenous wild-type protein from which it is derived.

The phrase “VEGF binding antagonist” refers to a functional class ofagents that bind to VEGF-A or its receptor, VEGFR-2, so that, as aresult of the binding, activation of VEGFR-2 by VEGF-A is blocked orinhibited. As used herein, the term “VEGF binding antagonists” includeantibody fragments, peptide inhibitors, dominant negative peptides andsmall molecule drugs, either in isolated forms or as part of a fusionprotein or conjugate.

The phrase “Tie2 tyrosine kinase receptor binding antagonist” refers toa functional class of agents that bind to a Tie2 tyrosine kinasereceptor or one of its ligands so that, as a result of the binding,activation of the Tie2 tyrosine kinase receptor by one or more of itsligands (i.e., Ang1, Ang2, Ang3 and Ang4) is blocked or inhibited. Asused herein, the term “Tie2 tyrosine kinase receptor binding antagonist”include antibody fragments, peptide inhibitors, dominant negativepeptides and small molecule drugs, either in isolated forms or as partof a fusion protein or conjugate.

The phrase “wherein the one or more mutations reduce proteolyticcleavage of the antitumor antagonist” should be interpreted as relatingto an otherwise identical antagonist containing a wild-type ECD.

The phrase “small molecule drug” refers to a molecular entity, oftenorganic or organometallic, that is not a polymer, that has medicinalactivity, and that has a molecular weight less than about 2 kDa, lessthan about 1 kDa, less than about 900 Da, less than about 800 Da or lessthan about 700 Da. The term encompasses most medicinal compounds termed“drugs” other than protein or nucleic acids, although a small peptide ornucleic acid analog can be considered a small molecule drug. Examplesinclude chemotherapeutic anticancer drugs and enzymatic inhibitors.Small molecules drugs can be derived synthetically, semi-synthetically(i.e., from naturally occurring precursors), or biologically.

When describing polypeptide domain arrangements with hyphens betweenindividual domains (e.g., CH2-CH3), it should be understood that theorder of the listed domains is from the amino terminal end to thecarboxy terminal end.

The term “immunoconjugate” refers to antibody which is fused by covalentlinkage to an inhibitory peptide or small molecule drug. The peptide orsmall molecule drug can be linked to the C-terminus of a constant heavychain or to the N-terminus of a variable light and/or heavy chain.

A “linker” may be used to link the peptide or small molecule drug, suchas a maytansinoid, to the antitumor antagonists in a stable, covalentmanner. Linkers can be susceptible to or be substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Suitable linkers are well known in the art and include, for example,disulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Linkers alsoinclude GGGGS, charged linkers, and hydrophilic forms thereof asdescribed herein and know in the art. The immunoconjugate may furtherinclude a flexible 3-15 amino acid peptide (or spacer) between anantitumor antagonist and the peptide and/or small molecule drug. In someembodiments, the linker comprises 3, 4, 5, 6, 7 or 8 repeats of GGGGS.In some embodiments, the linker consists of 3 or 4 repeats of GGGGS.

As used herein, the term “scaffold”, refers to any polymer of aminoacids that exhibits properties desired to support the function ofantagonist, including addition of antibody specificity, enhancement ofantibody function or support of antibody structure and stability. Ascaffold can be grafted with binding domains of a donor polypeptide toconfer the binding specificity of the donor polypeptide onto thescaffold.

As used herein, the phrase “multispecific inhibitor” refers to amolecule comprising at least two targeting domains with differentbinding specificities. In some embodiments, the multispecific inhibitoris a polypeptide comprising a scaffold and two or more immunoglobulinantigen binding domains targeting different antigens or epitopes. Incertain embodiments, the multispecific inhibitor is a bispecificantibody or antagonist. In other embodiments, the multispecificinhibitor is a trispecific antibody or antagonist.

As used herein, the phrase “bispecific” refers to a molecule comprisingat least two targeting domains with different binding specificities.Each targeting domain is capable of binding specifically to a targetmolecule and inhibiting a biological function of the target moleculeupon binding to the target molecule. In some embodiments, the bispecificcheckpoint regulator antagonist is a polymeric molecule having two ormore peptides. In some embodiments, the targeting domain comprisesantigen binding domain or a CDR of antibody. In some embodiments, thebispecific inhibitor is a bispecific antibody.

The terms “bispecific antibody,” and “bispecific antagonist” are usedinterchangeably herein with reference to antibody that can specificallybind two different antigens (or epitopes). In some embodiments, thebispecific antibody is a full-length antibody that binds one antigen (orepitope) on one of its two binding arms (one pair of HC/LC), and binds adifferent antigen (or epitope) on its second arm (a different pair ofHC/LC). In these embodiments, the bispecific antibody has two distinctantigen binding arms (in both specificity and CDR sequences) and ismonovalent for each antigen it binds to.

In other embodiments, the bispecific antibody is a full-length antibodythat can bind two different antigens (or epitopes) in each of its twobinding arms (two pairs of HC/LC) In these embodiments, the bispecificantibody has two identical antigen binding arms, with identicalspecificity and identical CDR sequences, and is bivalent for eachantigen it binds to.

Exemplary bispecific antibodies may include asymmetric IgG-likeantibodies (e.g., triomab/quadroma, Trion Pharma/Fresenius Biotech);knobs-into-holes antibodies (Genentech); Cross MAbs (Roche);electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); strandexchange engineered domain (SEED) body (EMD Serono; biolonic, Merus);Fab-exchanged antibodies (Genmab), symmetric IgG-like antibodies (e.g.dual targeting (DT)-Ig (GSK/Domantis); two-in-one antibody (Genentech);crosslinked MAbs (Karmanos Cancer Center), mAb² (F-star); Cov X-body(Cov X/Pfizer); dual variable domain (DVD)-Ig fusions (Abbott); IgG-likebispecific antibodies (Eli Lilly); Ts2Ab (Medimmune/AZ); BsAb(ZymoGenetics); HERCULES (Biogen Idec,TvAb, Roche); scFv/Fc fusions;SCORPION (Emergent BioSolutions/Trubion, ZymoGenetics/BMS); dualaffinity retargeting technology (Fc-DART), MacroGenics; dual(scFv)₂-Fabs (National Research Center for Antibody Medicine); F(ab)₂fusions (Medarex/AMGEN); dual-action or Bis-Fab (Genentech);Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv- anddiabody-based antibodies (e.g., bispecific T cell engagers (BiTEs,Micromet); tandem diabodies (Tandab, Affimed); DARTs (MacroGenics);single-chain diabodies; TCR-like antibodies (AIT, Receptor Logics);human serum albumin scFv fusion (Merrimack); COMBODIES (Epigen Biotech);and IgG/non-IgG fusions (e.g., immunocytokines (EMDSerono, Philogen,ImmunGene, ImmunoMedics).

The terms “treat” and “treatment” refer to the amelioration of one ormore symptoms associated with a cell proliferative disorder; preventionor delay of the onset of one or more symptoms of a cell proliferativedisorder; and/or lessening of the severity or frequency of one or moresymptoms of cell proliferative disorder.

The phrases “to a patient in need thereof”, “to a patient in need oftreatment” or “a subject in need of treatment” includes subjects, suchas mammalian subjects, that would benefit from administration of theantitumor antagonist of the present disclosure for treatment of a cellproliferative disorder.

The terms “therapeutically effective amount”, “pharmacologicallyeffective amount”, and “physiologically effective amount” are usedinterchangeably to mean the amount of antitumor antagonist that isneeded to provide a threshold level of active antagonist agents in thebloodstream or in the target tissue. The precise amount will depend uponnumerous factors, e.g., the particular active agent, the components andphysical characteristics of the composition, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one skilled in the art, based upon the informationprovided herein or otherwise available in the relevant literature.

The terms, “improve”, “increase” or “reduce”, as used in this context,indicate values or parameters relative to a baseline measurement, suchas a measurement in the same individual prior to initiation of thetreatment described herein, or a measurement in a control individual (ormultiple control individuals) in the absence of the treatment describedherein.

A “control individual” is an individual afflicted with the same cellproliferative disorder as the individual being treated, who is about thesame age as the individual being treated (to ensure that the stages ofthe disease in the treated individual and the control individual(s) arecomparable). The individual (also referred to as “patient” or “subject”)being treated may be a fetus, infant, child, adolescent, or adult humanwith a cell proliferative disorder.

The term “cell proliferative disorder” refers to a disordercharacterized by abnormal proliferation of cells. A proliferativedisorder does not imply any limitation with respect to the rate of cellgrowth, but merely indicates loss of normal controls that affect growthand cell division. Thus, in some embodiments, cells of a proliferativedisorder can have the same cell division rates as normal cells but donot respond to signals that limit such growth. Within the ambit of “cellproliferative disorder” is a neoplasm, cancer or tumor.

The term “cancer” or “tumor” refers to any one of a variety of malignantneoplasms characterized by the proliferation of cells that have thecapability to invade surrounding tissue and/or metastasize to newcolonization sites, and includes leukemia, lymphoma, carcinoma,melanoma, sarcoma, germ cell tumor and blastoma. Exemplary cancers fortreatment with the methods of the instant disclosure include cancer ofthe brain, bladder, breast, cervix, colon, head and neck, kidney, lung,non-small cell lung, mesothelioma, ovary, prostate, stomach and uterus,leukemia, and medulloblastoma.

The term “leukemia” refers to progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Exemplary leukemias include, for example, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “carcinoma” refers to the malignant growth of epithelial cellstending to infiltrate the surrounding tissues and give rise tometastases. Exemplary carcinomas include, for example, acinar carcinoma,acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma,carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma,alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare,basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolarcarcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriformcarcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloidcarcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma,carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma,cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonalcarcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinomaepitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giantcell carcinoma, carcinoma gigantocellulare, glandular carcinoma,granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

The term “sarcoma” refers to a tumor made up of a substance like theembryonic connective tissue and is generally composed of closely packedcells embedded in a fibrillar or homogeneous substance. Exemplarysarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphomas (e.g., Non-Hodgkin Lymphoma), immunoblastic sarcoma ofT-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parostealsarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma,synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” refers to a tumor arising from the melanocyticsystem of the skin and other organs. Melanomas include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's Disease, multiplemyeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid,premalignant skin lesions, testicular cancer, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, and adrenal corticalcancer.

I. Bispecific Antagonists Targeting the TGF-β Pathway

TGF-β is considered the primary cytokine mediating immunosuppressionthrough the induction and maintenance of T regulatory cells, as well asthe direct suppression of innate and adaptive immune cells such as NK,DC, and T cells. TGF-β also plays roles in neoangiogenesis andstabilizing tumor vessels, as well as directly impacting tumors throughepithelial mesenchymal transition leading to cell migration andinvasion. As a potent inducer of angiogenesis, TGF-β1 provides acritical support system for solid tumors and plays an important role intumor cell dissemination. Accordingly, TGF-β status is a strongpredictor of anti-PD1/PD-L1 resistance and overall survival for avariety of cancers.

Many cells synthesize TGF-β and almost all of them have specificreceptors for these peptides. TGF-β 1, TGF-β2, and TGF-β3 all functionthrough the same receptor signaling system. The active form of TGF-β isa dimer that signals through the formation of a membrane boundheterotetramer composed of the serine threonine type 1 and type 2receptors, TGF-β RI and TGF RII, respectively. Recently, TGF-β pathwayinhibitors have been developed in the form of e.g., antibodies orbinding fragments directed against TGF-β1 or TGF-β1 RII, such asdominant negative fusion protein fragments containing the extracellulardomain (ECD) of TGF-β1 RII.

However, in the course of developing such binding reagents, theinventors of the present application have discovered that antagonistsbearing a TGFβ1 RII ECD domain exhibit unacceptable levels ofproteolytic degradation or clipping over time. To address this problem,TGF-β1 RII ECD mutant variants have been developed that reduce clippingwithin this domain. As further described herein, the TGFβ1 RII ECDs havebeen further combined with additional binding agents targetingadditional checkpoint regulator pathways or angiogenesis pathways togenerate bispecific antagonists with significantly reduced level ofproteolytic degradation or clipping over time.

In one aspect, the present application provides a bispecific antagonistinhibiting both the TGFβ pathway and the PD-1/PD-L1 checkpoint regulatorpathway.

In another aspect, the present application provides a bispecificantagonist inhibiting both the TGFβ pathway and the vascular endothelialgrowth factor (VEGF) pathway, vascular endothelial growth factorreceptor (VEGFR), or both.

A. Bispecific Antagonists Targeting the TGF-β and PD-1/PD-L1 Pathways

In some embodiments, the bispecific antagonists include a firsttargeting domain specifically inhibiting the TGF-β pathway and a secondtargeting domain specifically binding PD-1 or PD-L1. In someembodiments, the first targeting domain comprises a TGF-β pathwayinhibitor.

TGF-β Pathway Inhibitors

The TGF-β pathway involves a multifunctional set of peptides thatcontrol cell proliferation and differentiation, migration and adhesion,extracellular matrix modification including tumor stroma andimmunosuppression, angiogenesis and desmoplasia, apoptosis, and otherfunctions in many cell types. TGF-β is considered the primary cytokinemediating immunosuppression through the induction and maintenance of Tregulatory cells, as well as the direct suppression of innate andadaptive immune cells such as NK, DC, and T cells. TGFB also plays rolesin neoangiogenesis and stabilizing tumor vessels, as well as directlyimpacting tumors through epithelial mesenchymal transition leading tocell migration and invasion. As a potent inducer of angiogenesis, TGF-β1provides a critical support system for solid tumors, as well as amechanism for tumor cell dissemination.

TGF-β status is a strong predictor of anti-PD1/PD-L1 resistance andoverall survival for a variety of cancers. For example, high TGF-β1levels are associated with poor response to PD1/PD-L1 inhibition inmetastatic urothelial cancer patients (Nature (2018) 554(7693):544-548),and a high TGFβ signature is associated with poor prognosis across 33different cancer types (Immunity (2018) 48:812-830). Inhibition of TGFβwith anti-PD1/PD-L1 further releases CD8 effector cells to kill tumorcells, as well as stimulate other cell types to increase tumor killingcapacity.

Many cells synthesize TGF-β and almost all of them have specificreceptors for these peptides. TGF-β1, TGF-β2, and TGF-β3 all functionthrough the same receptor signaling system. The active form of TGF-β isa dimer that signals through the formation of a membrane boundheterotetramer composed of the serine threonine type 1 and type 2receptors, TGF-β RI and TGFβ RII, respectively.

As used herein, a TGF-β pathway inhibitor may be in the form of e.g.,antibodies or variable domain fragments directed against TGF-β1 or aTGF-β1 RII, a TGF binding peptide, or dominant negative fusion proteinfragment, such as the extracellular domain (ECD) of TGF-β1 RII.

TGF-β1 RII ECD and Variants

In some embodiments, the TGF-β pathway inhibitor comprises a TGF-β1 RIIECD. An exemplary human TGF-β1 RII ECD (wild-type) has the amino acidsequence set forth in SEQ ID NO: 89. The inventors of the presentapplication have discovered that antagonists bearing a TGFβ1 RII ECDdomain exhibit unacceptable levels of proteolytic degradation orclipping over time upon storage as described in e.g., Example 9.Therefore, in some embodiments, the TGF-β pathway inhibitors of thepresent application comprises variants of the human TGF-β1 RII ECD(hereinafter “TGFBR variants”), that contain one or more modificationsthat reduce proteolytic degradation or clipping. In some embodiments,the modifications comprise substitutions of amino acid residues,deletion of amino acid residues, insertion of amino acid residues, or acombination thereof. In some embodiments, the TGFBR variants compriseone or more substitution mutations at positions 6, 7, 13, 16, 17, 20,22, and/or 23 of SEQ ID NO: 89. In some embodiments, the one or moresubstitution mutations replace a non-polar amino acid residue with apolar or hydrophobic amino acid residue.

In some embodiments, the TGFBR variants comprise one or more deletionsof amino acids relative to the wide type human TGF-β1 RII ECD sequences.In some embodiments, the one or more deletions are directed to a regionencompassing amino acid residues 1-20 of SEQ ID NO: 89 and may includeany of the residues in therein. In more particular embodiments, the oneor more mutations include a deletion of amino acid residues 1-7, 1-12,1-13, 1-15, 1-20, 7-12, 7-13, 7-15, 7-20, 8-20, 9-20, 10-20, 11-20,12-20, 13-20, 14-20, 15-20, 16-20 and/or 17-20 of SEQ ID NO: 89. In moreparticular embodiments, the one or more deletions include amino acidresidues 6, 7, 12, 13, 15, 16, 17 or 20 of SEQ ID NO: 89.

In some embodiments, the TGFBR variant comprises (1) a deletion of oneor more amino acid residues in the region of amino acid residues 1-20 ofSEQ ID NO: 89 and one or more substitutions at amino acid residues 6, 7,13, 16, 17, 20, 22, and/or 23 of SEQ ID NO: 89. In some embodiments, theTGFBR variant comprises (1) a deletion of amino acid residues 1-7 of SEQID NO: 89 and (2) an amino acid substitution at amino acid residue 13,16, 17, 20, 22, and 23 of SEQ ID NO: 89 or two amino acid substitutionsat amino acid residues 16 and 17 of SEQ ID NO: 89. In some embodiments,one or more amino acid residues 6, 7, 13, 16, 17, 20, 22, and/or 23 ofSEQ ID NO: 89 are replaced with one or more polar amino acid residues.In some embodiments, one or more amino acid residues 6, 7, 13, 16, 17,20, 22, and/or 23 of SEQ ID NO: 89 are replaced with one or morehydrophobic amino acid residues.

In some embodiments, the TGFBR variant comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 114-123 and 251-270(FIGS. 41A-41C).

In some embodiments, the TGFBR variant comprises an immunoglobulin heavychain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 124-133, 148, 150, and 271-290 (FIGS. 42A-42H,44A).

In other embodiments, the TGFBR variant comprises an immunoglobulinheavy chain comprising the amino acid sequence of SEQ ID NOS: 145, SEQID NO: 147, or SEQ ID NO: 303 (FIG. 43).

In other embodiments, the TGFBR variant comprises an immunoglobulinheavy chain comprising the amino acid sequence of SEQ ID NOS: 148, SEQID NO: 150, or SEQ ID NOS: 291-302 (FIGS. 44A-44D).

It should be understood that wherever a TGFBR variant is cited in thisapplication as comprising an immunoglobulin heavy chain comprising anamino acid sequence selected from the group consisting of SEQ ID NOS:124-133, 145, 147, 148, 150 and 271-303 (herein “the particular aminoacid sequence”), the embodiment should be further construed asrepresenting additional heavy chain embodiments characterized by allheavy chain sequence combinations derived therefrom in which one or moreof the pre-existing anti-PD1 amino acid sequences, including HCDR1,HCDR2 and HCDR3 sequences as set forth in FIG. 1 or one or more of thepre-existing anti-PD-L1 sequences, including HCDR1, HCDR2 and HCDR3sequences as set forth in FIG. 3 are substituted in place of thepre-existing anti-PD1 CDR sequences or anti-PD-L1 CDR sequences presentin “the particular amino acid sequence.”

Similarly, with respect to the preceding paragraph, any of theimmunoglobulin light chains described as pairing with the aforementionedheavy chains represented by SEQ ID NOS: 124-133, 145, 147, 148, 150 and271-303 should be further construed as representing additional lightchain embodiments characterized by all light chain sequence combinationsderived therefrom in which one or more of the pre-existing anti-PD1amino acid sequences, including LCDR1, LCDR2 and LCDR3 sequences as setforth in FIG. 1 or one or more of the pre-existing anti-PD-L1 sequences,including LCDR1, LCDR2 and LCDR3 sequences as set forth in FIG. 3 aresubstituted in place of the pre-existing anti-PD1 CDR sequences oranti-PD-L1 CDR sequences present in “the particular amino acidsequence.”

Likewise, it should be understood that wherever a TGFBR variant is citedin this application as comprising an immunoglobulin heavy chaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 124-133, 145, 147, 148, 150 and 271-303 (“the particularamino acid sequence”), the embodiment should be further construed asrepresenting additional heavy chain embodiments, including all lightchain sequence combinations derived therefrom in which the pre-existinganti-PD1 heavy chain variable region (HCVR) amino acid sequence in the“particular amino acid sequence” is substituted for a different anti-PD1HCVR selected from the group consisting of SEQ ID NOS: 29, 31, 33, 35,37 and 39 as shown in FIGS. 2A-2B or in which the pre-existinganti-PD-L1 HCVR amino acid sequence in “the particular amino acidsequence” is substituted for a different anti-PD-L1 HCVR selected fromthe group consisting of SEQ ID NOS: 72, 74, 76, 78, 80, 82, 84 and 86 asshown in FIGS. 4A-4C.

Similarly, with respect to the preceding paragraph, any of theimmunoglobulin light chains described as pairing with the aforementionedheavy chains in “the particular amino acid sequence” should be furtherconstrued as representing additional light chain embodiments, includingall light chain sequence combinations derived from the aforementionedlight chain sequences in which the pre-existing anti-PD1 light chainvariable region (LCVR) amino acid sequence in “the particular amino acidsequence” is substituted for a different anti-PD1 LCVR selected from thegroup consisting of SEQ ID NOS: 30, 32, 34, 36, 38 and 40 as shown inFIGS. 2A-2B or in which the pre-existing anti-PD-L1 LCVR amino acidsequence in “the particular amino acid sequence” is substituted for adifferent anti-PD-L1 LCVR selected from the group consisting of SEQ IDNOS: 73, 75, 77, 79, 81, 83, 85 and 87 as shown in FIGS. 4A-4C.

In some embodiments, the TGF-β pathway inhibitor is fused to thecarboxy-terminus of an IgG in a bispecific antitumor antagonist, asdepicted in e.g., FIGS. 5A-5B, 8A-8C and 20A-20B. Alternatively, theTGF-β1 RII ECD may be fused to the amino-terminus of an IgG in abispecific antitumor antagonist. In yet other embodiments, the TGF-β1RII ECD is inserted within the IgG Fc receptor (i.e., CH2 or CH3regions) of an IgG as depicted in e.g., FIG. 5B.

In some embodiments, anti-TGF-β1, anti-TGF-β1 RII antibodies or variableregion fragments thereof may be used in place of a TGF-β1 RII ECD.Exemplary anti-TGF-β1 antibodies are described in U.S. Pat. Nos.7,067,637, 7,494,651, 7,527,791, and 7,619,069. Exemplary anti-TGF-β1RII antibodies are described in U.S. Pat. No. 7,579,186. Alternatively,a TGF-β1 RII ECD may be substituted with one or more anti-TGF-β1 and/oranti-TGF-β1 RII peptide inhibitors. An exemplary TGF-β1 peptideinhibitor is KRIWFIPRSSWYERA (SEQ ID NO: 155).

Anti-PD-1 Antibody and Anti-PD-1 Antibody Fragments

In some embodiments, the bispecific TGFβ pathway antagonist includes ananti-PD-1 antibody or antibody fragment. In one embodiment, the PD-1inhibitor for use in the present application is antibody, orantigen-binding portion thereof, comprising: an immunoglobulin heavychain complementarity determining region 1 (CDR1) sequence selected fromthe group consisting of SEQ ID NOS: 1, 4, 7, 9, and 12; animmunoglobulin heavy chain CDR2 sequence selected from the groupconsisting of SEQ ID NOS: 2, 5, 10, and 13; an immunoglobulin heavychain CDR3 sequence selected from the group consisting of SEQ ID NOS: 3,6, 8, 11, and 14; an immunoglobulin light chain CDR1 sequence selectedfrom the group consisting of SEQ ID NOS: 15, 18, 21-23, and 26; animmunoglobulin light chain CDR2 sequence selected from the groupconsisting of SEQ ID NOS: 16, 19, 24, and 27; or an immunoglobulin lightchain CDR3 sequence selected from the group consisting of SEQ ID NOS:17, 20, 25, and 28.

In another embodiment, the PD-1 inhibitor for use in the presentapplication (as e.g., the second targeting domain) is antibody, orantigen-binding portion thereof, comprising: an immunoglobulin heavychain CDR1 sequence selected from the group consisting of SEQ ID NOS: 1,4, 7, 9, and 12; an immunoglobulin heavy chain CDR2 sequence selectedfrom the group consisting of SEQ ID NOS: 2, 5, 10, and 13; animmunoglobulin heavy chain CDR3 sequence from the group consisting ofSEQ ID NOS: 3, 6, 8, 11, and 14; an immunoglobulin light chain CDR1sequence selected from the group consisting of SEQ ID NOS: 15, 18,21-23, and 26; an immunoglobulin light chain CDR2 sequence selected fromthe group consisting of SEQ ID NOS: 16, 19, 24, and 27; and animmunoglobulin light chain CDR3 sequence selected from the groupconsisting of SEQ ID NOS: 17, 20, 25, and 28.

In another embodiment, the PD-1 inhibitor for use in the presentapplication includes: an immunoglobulin heavy chain variable region(HCVR) having at least 80%, 85%, 90%, 95%, or 99% identity to an HCVRamino acid sequence selected from the group consisting of SEQ ID NOS:29, 31, 33, 35, 37, and 39; an immunoglobulin light chain variableregion (LCVR) having at least 80%, 85%, 90%, 95%, or 99% identity to anLCVR amino acid sequence selected from the group consisting of SEQ IDNOS: 30, 32, 34, 36, 38, and 40; or both.

In another embodiment, the PD-1 inhibitor for use in the presentapplication includes: an HCVR amino acid sequence selected from thegroup consisting of SEQ ID NOS: 29, 31, 33, 35, 37, and 39; an LCVRamino acid sequence selected from the group consisting of SEQ ID NOS:30, 32, 34, 36, 38, and 40; or both.

In one embodiment, the PD-1 inhibitor includes: an HCVR having at least80%, 85%, 90%, 95%, or 99% identity to the HCVR amino acid sequence ofSEQ ID NO: 39; an LCVR having at least 80%, 85%, 90%, 95%, or 99%identity to the LCVR amino acid sequence of SEQ ID NO: 40; or both. In amore particular embodiment, the PD-1 inhibitor has an HCVR sequence ofSEQ ID NO: 39; an LCVR amino acid sequence of SEQ ID NO: 40; or both.

In another embodiments, the PD-1 inhibitor has an immunoglobulin heavychain variable region that comprises (1) an HCDR1 of SEQ ID NO: 12, anHCDR2 of SEQ ID NO: 13 and an HCDR 3 of SEQ ID NO: 14 and (2) an HFR1having at least 80%, 85% or 90% identity to the amino acid sequence ofSEQ ID NO: 196, an HFR2 having at least 80%, 85% or 90% identity to theamino acid sequence of SEQ ID NO: 190, an HFR3 having at least 80%, 85%or 90% identity to the amino acid sequence of SEQ ID NO: 201, an HFR4having at least 80%, 85% or 90% identity to the amino acid sequence ofSEQ ID NO: 187, and an immunoglobulin heavy chain variable region thatcomprises (1) an LCDR1 of SEQ ID NO: 26, an LCDR2 of SEQ ID NO: 27 andan LCDR 3 of SEQ ID NO: 28 and (2) an LFR1 having at least 80%, 85% or90% identity to the amino acid sequence of SEQ ID NO: 202, an LFR2having at least 80%, 85% or 90% identity to the amino acid sequence ofSEQ ID NO: 203, an LFR3 having at least 80%, 85% or 90% identity to theamino acid sequence of SEQ ID NO: 204, an LFR4 having at least 80%, 85%or 90% identity to the amino acid sequence of SEQ ID NO: 205.

In another embodiment, the TGF-β1 antitumor antagonist includes a PD-1inhibitor comprising: an immunoglobulin heavy chain having at least 80%,85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:92 or SEQ ID NO: 113; a light chain having at least 80%, 85%, 90%, 95%,or 99% identity to the amino acid sequence of SEQ ID NO: 93; or both. Ina more particular embodiment, the TGF-β1 antitumor antagonist includes aPD-1 inhibitor comprising: an immunoglobulin heavy chain having theamino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 113; animmunoglobulin light chain having the amino acid sequence of SEQ ID NO:93; or both.

In another embodiment, the TGF-β1 antitumor antagonist includes a PD-1inhibitor comprising: an immunoglobulin heavy chain having at least 80%,85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:94 or SEQ ID NO: 172; an immunoglobulin light chain having at least 80%,85%, 90%, 95%, or 99% identity to the LCVR amino acid sequence of SEQ IDNO: 95; or both. In a more particular embodiment, the TGF-1 antitumorantagonist includes a PD-1 inhibitor comprising: an immunoglobulin heavychain having the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 172;an immunoglobulin light chain having the amino acid sequence of SEQ IDNO: 95; or both. In another embodiment, the PD-1/TGF-β1 antitumorantagonist includes a TGF-β1 RII ECD inserted within a CH3 loop asdepicted in FIG. 5B.

In one embodiment, the TGF-β1 antitumor antagonist includes a PD-1inhibitor comprising: a heavy chain having at least 80%, 85%, 90%, 95%,or 99% identity to the amino acid sequence of SEQ ID NO: 151; a lightchain having at least 80%, 85%, 90%, 95%, or 99% identity to the aminoacid sequence of SEQ ID NO: 152; or both. In a more particularembodiment, the TGF-β1 antitumor antagonist includes a PD-1 inhibitorcomprising: an immunoglobulin heavy chain having the amino acid sequenceof SEQ ID NO: 151; immunoglobulin light chain having the amino acidsequence of SEQ ID NO: 152, or both.

Other PD-1 targeted antagonists include anti-PD-1 antibodies, such asnivolumab (BMS-936558, MDX-1106, OPDIVO™), a human immunoglobulin G4(IgG4) mAb (Bristol-Myers Squibb); pembrolizumab (MK-3475,lambrolizumab, KEYTRUDA™), a humanized immunoglobulin G4 (IgG4) mAb(Merck); pidilizumab (CT-011)(Medivation); and AMP-224 (Merck).Anti-PD-1 antibodies are commercially available, for example from ABCAM(AB137132), BIOLEGEND™ (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE(J105, J116, MIH4). Anti-PD-1 targeted antitumor antagonists may includeany of the HCVRs, LCVRs, and/or CDRs derived from any of the anti-PD-1antibodies described herein, including those described in FIGS. 1-2.

Anti-PD-L1 Antibody and Anti-PD-L1 Antibody Fragments

In some embodiments, the bispecific TGF-β1 pathway antagonist furtherincludes an anti-PD-L1 antibody or antibody fragment. In one embodiment,the PD-L1 inhibitor for use in the present application is antibody, orantigen-binding portion thereof, comprising: an immunoglobulin heavychain CDR1 sequence selected from the group consisting of SEQ ID NOS:41, 44, 50, and 53; an immunoglobulin heavy chain CDR2 sequence selectedfrom the group consisting of SEQ ID NOS: 42, 45, 47, 49, 51, and 54; animmunoglobulin heavy chain CDR3 sequence selected from the groupconsisting of SEQ ID NOS: 43, 46, 48, 52, and 55; an immunoglobulinlight chain CDR1 sequence selected from the group consisting of SEQ IDNOS: 56, 59, 63, 66, and 69; an immunoglobulin light chain CDR2 sequenceselected from the group consisting of SEQ ID NOS: 57, 60, 64, 67, and70; or an immunoglobulin light chain CDR3 sequence selected from thegroup consisting of SEQ ID NOS: 58, 61, 62, 65, 68, and 71.

In another embodiment, the PD-L1 inhibitor for use in the presentapplication (as e.g., the second targeting domain) is antibody, orantigen-binding portion thereof, comprising: an immunoglobulin heavychain CDR1 sequence selected from the group consisting of SEQ ID NOS:41, 44, 50, and 53; an immunoglobulin heavy chain CDR2 sequence selectedfrom the group consisting of SEQ ID NOS: 42, 45, 47, 49, 51, and 54; animmunoglobulin heavy chain CDR3 sequence selected from the groupconsisting of SEQ ID NOS: 43, 46, 48, 52, and 55; an immunoglobulinlight chain CDR1 sequence selected from the group consisting of SEQ IDNOS: 56, 59, 63, 66, and 69; an immunoglobulin light chain CDR2 sequenceselected from the group consisting of SEQ ID NOS: 57, 60, 64, 67, and70; and an immunoglobulin light chain CDR3 sequence selected from thegroup consisting of SEQ ID NOS: 58, 61, 62, 65, 68, and 71.

In another embodiment, the PD-L1 inhibitor for use in the presentapplication includes: an immunoglobulin HCVR having at least 80%, 85%,90%, 95%, or 99% identity to an HCVR amino acid sequence selected fromthe group consisting of SEQ ID NOS: 72, 74, 76, 78, 80, 82, 84, and 86;an immunoglobulin LCVR having at least 80%, 85%, 90%, 95%, or 99%identity to an LCVR amino acid sequence selected from the groupconsisting of SEQ ID NOS: 73, 75, 77, 79, 81, 83, 85, and 87; or both.In another embodiment, the PD-L1 inhibitor for use in the presentapplication includes: an HCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 72, 74, 76, 78, 80, 82, 84, and86; an LCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 73, 75, 77, 79, 81, 83, 85, and 87; or both.

In one embodiment, the PD-L1 inhibitor includes: an HCVR having at least80%, 85%, 90%, 95%, or 99% identity to the HCVR amino acid sequence ofSEQ ID NO: 86; an LCVR having at least 80%, 85%, 90%, 95%, or 99%identity to the LCVR amino acid sequence of SEQ ID NO: 87; or both. In amore particular embodiment, the PD-L1 inhibitor includes: an HCVR havingthe amino acid sequence of SEQ ID NO: 86; an LCVR having the amino acidsequence of SEQ ID NO: 87; or both.

In another embodiment, the TGF-β1 antitumor antagonist includes a PD-L1inhibitor comprising: an immunoglobulin heavy chain having at least 80%,85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:94 or SEQ ID NO: 172; an immunoglobulin light chain having at least 80%,85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:95; or both. In a more particular embodiment, the TGF-β1 antitumorantagonist includes a PD-L1 inhibitor comprising: an immunoglobulinheavy chain having the amino acid sequence of SEQ ID NO: 94 or SEQ IDNO: 172; an immunoglobulin light chain having the amino acid sequence ofSEQ ID NO: 95; or both.

In another embodiment, the PD-L1/TGF-β1 antitumor antagonist includes aTGF-β1 RII ECD inserted within a CH3 loop as depicted in FIG. 5B.

In a particular embodiment, the TGF-β1 antitumor antagonist includes aPD-L1 inhibitor comprising: an immunoglobulin heavy chain having atleast 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence ofSEQ ID NO: 153; an immunoglobulin light chain having at least 80%, 85%,90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 154;or both. In a more particular embodiment, the TGF-β1 antitumorantagonist includes a PD-L1 inhibitor comprising: an immunoglobulinheavy chain having the amino acid sequence of SEQ ID NO: 153; animmunoglobulin light chain having the amino acid sequence of SEQ ID NO:154, or both.

Other anti-PD-L1 targeted antagonists include anti-PD-L1 antibodies,such as atezolizumab (MPDL3280A, RG7446, Tecentriq), a fully humanizedIgG1 mAb (Genentech/Roche); BMS-936559 (MDX-1105), a fully human IgG4mAb (Bristol-Myers Squibb); durvalumab (MEDI4736, Imfinzi), a human IgGantibody (Medimmune/AstraZeneca); and avelumab (MSB0010718C, Bavencio),a fully human IgG4 monoclonal antibody (Merck, EMD Serono). Anti-PD-L1targeted antitumor antagonists may include any of the HCVRs, LCVRs,and/or CDRs derived from any of the anti-PD-L1 antibodies describedherein, including those described in FIGS. 3-4.

B. Bispecific Antagonists Targeting TGF-β1 and Angiogenesis Pathway(s)

In another aspect, a bispecific antitumor antagonist of the presentapplication includes a first targeting domain specifically binding TGFβ1or TGFβ1 RII, and a second targeting domain specifically binding VEGF-A,VEGFR, Ang1, Ang2, Tie2R, or a combination thereof. In theseembodiments, any of the above described TGFβ1- or TGFβ1 RII-bindingfragments may be used in combination with the second targeting domain.

Angiogenesis Pathways

Angiogenesis, the development of new blood vessels from pre-existingvessels, is essential for tumor growth and metastasis. Angiogenesisinhibition presents a potentially valuable strategy for treatingdiseases, such as cancer, in which progression (e.g., metastasis) isdependent on neovascularization. Inhibition of angiogenesis leads totumor cell death, which may feed tumor antigen into host antigenpresentation pathways. Angiogenesis pathway inhibitors may be in theform of e.g., antibodies, variable domain fragments, or dominantnegative fusion protein fragments.

1. VEGF/VEGFR Pathway

The principal VEGF pathway is mediated by the transmembrane tyrosinekinase VEGF-R2. Various isoforms of VEGF, particularly VEGF-A, bind toVEGF-R2, resulting in dimerization and activation throughphosphorylation of various downstream tyrosine kinases.

In some embodiments, the antagonist of the present application include aVEGF pathway antagonist that binds to VEGF-A or its receptor VEGFR-2 sothat, as a result of the binding, activation of VEGFR-2 by VEGF-A isblocked or inhibited.

In one embodiment, the TGFβ pathway antagonist further includes VEGFpathway antagonist in the form of a dominant negative VEGFR antagonistcorresponding to the extracellular domain (ECD) of human VEGF receptor 1or 2. In a particular embodiment, the TGFβ pathway antagonist includesaflibercept (also known as Zaltrap), a recombinant fusion proteincontaining VEGF-A binding portions from the extracellular domains ofhuman VEGF receptors 1 and 2 fused to the human IgG1 Fc portion. VEGFRECDs, such as aflibercept act as soluble receptor decoys for VEGF-A.

In one embodiment, the bispecific antitumor antagonist includes a firsttargeting domain comprising a TGFβ pathway inhibitor, and a secondtargeting domain specifically binding VEGF-A, wherein the secondtargeting domain comprises aflibercept. A suitable source of afliberceptcomprises the amino acid sequence set forth in SEQ ID NO: 88. In someembodiments, a bispecific antitumor antagonist includes a modifiedimmunoglobulin heavy chain comprising an amino terminal afliberceptdomain linked to a TGF-β1 RII ECD or a TGF-β1 RII ECD variant at thecarboxy-terminal end via an IgG1 or IgG4 Fc receptor (Bi-ZB-1) as shownin FIG. 8C. In one embodiment, the modified immunoglobulin heavy chainhas the amino acid sequence of SEQ ID NO: 105. Alternatively, the TGF-β1RII ECD or TGF-β1 RII ECD variant may be positioned at the aminoterminal end and the aflibercept domain may be positioned at thecarboxy-terminal end.

In another embodiment, the TGFβ pathway antagonist further includes VEGFpathway antagonist comprising anti-VEGFA or anti-VEGFR2 variable regionsequences, such as those derived from bevacizumab. Bevacizumab(AVASTIN™) is a humanized antibody comprising a human IgG1 frameworkregions (FRs) and antigen-binding complementarity-determining regionsfrom the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocksbinding of human VEGF-A to VEGFR1 and VEGFR-2. Approximately 93% of theamino acid sequence of bevacizumab, including most of the frameworkregions, is derived from human IgG1, and about 7% of the sequence isderived from the murine antibody A4.6.1. Bevacizumab has a molecularmass of about 149,000 Daltons and is glycosylated. In one embodiment, anHCVR of bevacizumab for use in the present application has the aminoacid sequence set forth in SEQ ID NO: 90, and an LCVR of bevacizumab hasthe amino acid sequence set forth in SEQ ID NO: 91.

In some embodiments, amino acid substitutions may be included in abevacizumab/AVASTIN antibody or fragment thereof as described in U.S.Pat. No. 7,575,893. Exemplary amino acid substitutions include, but arenot limited to E1Q, E6Q, L11V, Q13K, L18V, R19K, A23K, or combinationsthereof. In one embodiment, a mutant HCVR of bevacizumab for use in thepresent application has the amino acid sequence set forth in SEQ ID NO:96, which may be used in conjunction with the LCVR in SEQ ID NO: 91.

In one embodiment, the bispecific antitumor antagonist includes a firsttargeting domain specifically binding TGFβ1, such as a TGFβ1 RII ECD (orcontaining anti-TGF β1- or anti-TGFβ1 RII variable domains), and asecond targeting domain specifically binding VEGFA or VEGFR2, such asbevacizumab or any other anti-VEGF or anti-VEGFR2 antibodies, variableregion fragments thereof, or functionally active mutant fragmentsthereof.

In a particular embodiment, a bispecific antitumor TGF-β/VEGF-VEGFR2antagonist includes an immunoglobulin heavy chain having the amino acidsequence of SEQ ID NO: 102, an immunoglobulin light chain having theamino acid sequence of SEQ ID NO: 103, or both, such as Bi-AB-1 (FIG.8A), which contains wild-type bevacizumab variable region sequences.

In another embodiment, a bispecific antitumor TGF-β antagonist furtherincludes an immunoglobulin heavy chain having the amino acid sequence ofSEQ ID NO: 104, an immunoglobulin light chain having the amino acidsequence of SEQ ID NO: 103, or both, such as Bi-A1B-1 (FIG. 8B), whichcontains a mutant bevacizumab variable region sequence.

Additional anti-VEGF or anti-VEGFR antibodies or fragments thereof maybe based on ranibizumab (trade name Lucentis™), a monoclonal antibodyfragment derived from the same parent murine antibody as bevacizumab;the G6 or B20 series antibodies (e.g., G6-23, G6-31, B20-4.1) describedin U.S. Patent Publication Nos. 2006/0280747, 2007/0141065 and/or2007/0020267, as well as the antibodies described in U.S. Pat. Nos.7,297,334, 7,060,269, 6,884,879, 6,582,959, 6,703,020; 6,054,297; U.S.Patent Publication Nos. 2007/059312, 2006/009360, 2005/0186208,2003/0206899, 2003/0190317, and 2003/0203409.

An exemplary anti-VEGFR-2 antibody antagonist is the humanized IgG1monoclonal antibody, Ramucirumab, which binds to the extracellulardomain of VEGFR-2, thereby blocking its interaction with VEGF-A.Additional anti-VEGFR-2 antibodies are described in U.S. Pat. Nos.7,498,414, 6,448,077 and 6,365,157.

In some embodiments, the antitumor antagonists may further include oneor more small molecule antagonists of the VEGF pathway, such asmultikinase inhibitors of VEGFR-2, including sunitinib, sorafenib,cediranib, pazonpanib and nintedanib.

2. Ang-Tie2R Pathway

In some embodiments, the bispecific antagonist includes a targetingdomain containing at least one Tie2 receptor binding antagonist. A Tie2tyrosine kinase receptor binding antagonist binds to the Tie2 tyrosinekinase receptor or one of its ligands (i.e., Ang1, Ang2, Ang3 and Ang4)so that, as a result of the binding, activation of the Tie2 tyrosinekinase receptor by one or more of its ligands is blocked or inhibited.Like VEGF, angiopoietin 2 (Ang2) is a critical player in tumorangiogenesis. ANG2 and VEGF also work together to prevent antigenpresentation in dendritic cells and macrophages, enhance Tregaccumulation, and suppress Teff accumulation. It has been previouslyshown that inhibiting both VEGF and Ang2 improves survival in a mousebreast cancer model, Tg MMTV-PyMT. Moreover, the addition of anti-PD1further improves responses.

Tie2 tyrosine kinase receptor binding antagonists for use in the presentapplication may include antibody fragments, peptide inhibitors, dominantnegative peptides and small molecule drugs, either in isolated forms oras part of a fusion protein or conjugate. In one embodiment, the Tie2receptor binding antagonist is an inhibitory peptide from trebananib,TBN-P. In a specific embodiment, the inhibitory peptide comprises theamino acid sequence:AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 157).In another embodiment, the Tie2 receptor binding antagonist comprisesSEQ ID NO: 158 (TBN-P-IgG).

Other peptide inhibitors of Tie2 activation (including Ang-2 inhibitors)for use in the present application include A-11 (Compugen), whichcomprises the amino acid sequence ETFLSTNKLENQ (SEQ ID NO: 167); theCVX-060 peptide QK(Ac)YQPLDEK(Ac)DK(OP)TLYDQFMLQQG (SEQ ID NO: 168,Pfizer); the CVX-037 peptide (DFB)TNFMPMDDLEK(OP)RLYEQFILQQG (SEQ ID NO:169, Pfizer); and CGEN-25017 (Compugen). Additional peptide inhibitorsof Tie2 activation are described in U.S. Pat. No. 7,138,370.

Antibody inhibitors of Tie2 activation (and/or angiopoietin-2) for usein the present application include AMG-780 (Amgen), MEDI-3617(MedImmune/AstraZeneca), DX-2240 (Dyax/Sanofi-Aventis), REGN-910(Sanofi/Regeneron), RG7594 (Roche), LC06 (Roche), TAvi6 (Roche), AT-006(Roche/Affitech). Additional Tie2 receptor binding antibody antagonistsand antibody binding sequences therefrom are described in U.S. Pat. Nos.7,521,053, 7,658,924, and 8,030,025, as well as U.S. Patent PublicationNos. 2013/0078248, 2013/0259859, and 2015/0197578.

Tie2 binding antagonists for use in the present application may furtherinclude the small molecule inhibitors, CGI-1842 (CGI Pharmaceuticals),LP-590 (Locus Pharmaceuticals), ACTB-1003 (Act Biotech/Bayer AG),CEP-11981 (Cephalon/Teva), MGCD265 (Methylgene), Regorafenib (Bayer),Cabozantinib/XL-184/BMS-907351 (Exelixis), Foretnib (Exelixis), MGCD-265(MethylGene Inc.).

In certain particular embodiments, the bispecific checkpoint regulatorantagonist is a full-length antibody that binds human PD-1 or PD-L1 onone of its two binding arms (one pair of HC/LC), and binds a differentantigen (or epitope) on its second arm (a different pair of HC/LC). Inthese embodiments, the bispecific antibody has two distinct antigenbinding arms (in both specificity and CDR sequences), and is monovalentfor each antigen it binds to.

In some embodiments, the bispecific checkpoint regulator antagonist is afull-length antibody that can bind human PD-1 and/or PD-L1 in each ofits two binding arms (a pair of HC/LC). In these embodiments, thebispecific checkpoint regulator antagonist has two identical antigenbinding arms, with identical specificity and identical CDR sequences,and is bivalent for each antigen it binds to.

Immunoglobulin and Non-Immunoglobulin Scaffolds

The bispecific antitumor antagonists of the present application may beconstructed with an immunoglobulin backbone or non-immunoglobulinscaffold as described herein. In some embodiments, the bispecificantagonists of the present application are constructed with animmunoglobulin scaffold, such as an IgG1, IgG2 or IgG4 scaffoldcomprising CH1, CH2 and/or CH3 domains. Use of an IgG1 backbone ispreferable for cancer treatment where a target is present on antigenpresenting cells that can mediate antibody-dependent cell-mediatedcytotoxicity (ADCC). Use of an IgG4 backbone allows targeting of theantigen where antigen binding alone is sufficient to generate thedesired therapeutic benefits. IgG4-based antagonists precludeundesirable effector functions associated with e.g., IgG1 antibodies,including FcγR binding and complement activation.

Preferably, the first and second targeting domains are presented in ahumanized IgG1 or IgG4 scaffold. Further, the second targeting domainmay be fused to the carboxy-terminal end of an IgG1 or IgG4 scaffold.Additionally, the IgG1 or IgG4 scaffold may have a N297A or K447A aminoacid substitution. In some embodiments, the first targeting domain maycomprise one or more framework regions comprising one or more amino acidsubstitutions selected from the group consisting of E1Q, E6Q, L11V,Q13K, L18V, R19K, A23K, or any combination thereof. In otherembodiments, one or more amino acid residues in the IgG1 or IgG4scaffold are deglycosylated or mutated to produce a glycosylatedvariants thereof. Exemplary immunoglobulin scaffolds for use in thebispecific molecules described herein may be selected from the groupconsisting of SEQ ID NOS: 159-166 and 173-175.

Any one of the antibodies or antagonists can be configured in the formof a monoclonal antibody, chimeric antibody, humanized antibody, scFv,or multi-specific antibody. In addition, any of the antibody antagonistsdescribed herein may include multiple binding specificities targetingPD-1, PD-L1, VEGF, VEGFR, Angiopoietin, and/or Tie2R. Moreover, any ofthe antibody antagonists may be engineered to target multiple epitopesin a given target. Furthermore, in some embodiments, the checkpointantagonist and/or angiogenesis specificity may be included in the formof a dominant negative fusion protein, such as an extracellular domain(ECD) from a corresponding receptor.

The HCVRs and LCVRs described herein may be linked to anaturally-occurring Fc region or a non-naturally occurring or mutated Fcregion, e.g., an effectorless (IgG1 N297A/G) or mostly effectorless Fc(e.g., human IgG2 or IgG4) or, alternatively, an Fc with enhancedbinding to one or more activating Fc receptors (FcγRI, FcγRIIa orFcγRIIIa) so as to enhance T_(reg) depletion in the tumor environment.Accordingly, in certain embodiments the anti-PD-1, anti-PD-L1, and/oranti-VEGF HCVRs and LCVRs described herein may be linked to an Fccomprising one or more modifications, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, antibody described herein may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or it may be modified to alter its glycosylation, toalter one or more functional properties of the antibody. Morespecifically, in certain embodiments, the antibodies in the presentapplication may include modifications in the Fc region in order togenerate an Fc variant with (a) increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) increased ordecreased affinity for C1q and/or (d) increased or decreased affinityfor a Fc receptor relative to the parent Fc. Such Fc region variantswill generally comprise at least one amino acid modification in the Fcregion. Combining amino acid modifications is thought to be particularlydesirable. For example, the variant Fc region may include two, three,four, five, etc. substitutions therein, e.g., of the specific Fc regionpositions identified herein.

For uses where effector function is to be avoided altogether, e.g., whenantigen binding alone is sufficient to generate the desired therapeuticbenefit, and effector function only leads to (or increases the risk of)undesired side effects, IgG4 antibodies may be used, or antibodies orfragments lacking the Fc region or a substantial portion thereof can bedevised, or the Fc may be mutated to eliminate glycosylation altogether(e.g., N297A). Alternatively, a hybrid construct of human IgG2 (CH1domain and hinge region) and human IgG4 (CH2 and CH3 domains) may begenerated that is devoid of effector function, lacking the ability tobind FcγRs (like IgG2) and activate complement (like IgG4). When usingan IgG4 constant domain, it is usually preferable to include thesubstitution S228P, which mimics the hinge sequence in IgG1 and therebystabilizes IgG4 molecules, reducing Fab-arm exchange between thetherapeutic antibody and endogenous IgG4 in the patient being treated.

In certain embodiments, the anti-PD-1-, anti-PD-L1-, anti-VEGF-,anti-angiopoietin-, and/or or anti-Tie2R antibodies or fragments thereofmay be modified to increase its biological half-life. Various approachesmay be employed, including e.g., those that increase the bindingaffinity of the Fc region for FcRn. In one embodiment, the antibody isaltered within the CH1 or CL region to contain a salvage receptorbinding epitope taken from two loops of a CH2 domain of an Fc region ofan IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022. Thenumbering of residues in the Fc region is that of the EU index. Sequencevariants disclosed herein are provided with reference to the residuenumber followed by the amino acid that is substituted in place of thenaturally occurring amino acid, optionally preceded by the naturallyoccurring residue at that position. Where multiple amino acids may bepresent at a given position, e.g., if sequences differ between naturallyoccurring isotypes, or if multiple mutations may be substituted at theposition, they are separated by slashes (e.g., “X/Y/Z”).

Exemplary Fc variants that increase binding to FcRn and/or improvepharmacokinetic properties include substitutions at positions 259, 308,and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F,434Y, and 434M. Other variants that increase Fc binding to FcRn include:250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem.279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A(Shields et al. (2001) J. Biol. Chem., 276(9):6591-6604), 252F, 252Y,252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E,433K/434F/436H (Dall'Acqua et al. (2002) J. Immunol., 169:5171-5180,Dall'Acqua et al. (2006) J. Biol. Chem., 281:23514-23524, and U.S. Pat.No. 8,367,805.

Modification of certain conserved residues in IgG Fc (1253, H310, Q311,H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol.182:7663), have been proposed as a way to increase FcRn affinity, thusincreasing the half-life of the antibody in circulation (WO 98/023289).The combination Fc variant comprising M428L and N434S has been shown toincrease FcRn binding and increase serum half-life up to five-fold(Zalevsky et al. (2010) Nat. Biotechnol. 28:157). The combination Fcvariant comprising T307A, E380A and N434A modifications also extendshalf-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol.18:1759). In addition, combination Fc variants comprising M252Y-M428L,M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, andM428L-N434S variants have also been shown to extend half-life (U.S.Patent Publication No. 2006/173170). Further, a combination Fc variantcomprising M252Y, S254T and T256E was reported to increasehalf-life-nearly 4-fold. Dall'Acqua et al. (2006) J. Biol. Chem.281:23514.

Homodimers and Heterodimers

One of the challenges for efficiently producing bispecific antibodypreparations concerns mispairing of heavy and light chains whenco-expressing chains of different binding specificities. Table 1 listsseveral amino acid substitution options for overcoming mispairingbetween heavy chains of different binding specificities, which “enforce”or preferentially promote correct association between desired heavychains. Any approach to prevent or reduce mispairing between heavychains may be used to make the bispecific antitumor antagonistsaccording to the present disclosure.

The “knobs-into-hole” (KiH) approach relies on modifications of theinterface between the two CH3 domains where most interactions occur.Typically, a bulky residue is introduced into the CH3 domain of oneantibody heavy chain and acts similarly to a key. In the other heavychain, a “hole” is formed that is able to accommodate this bulkyresidue, mimicking a lock. The resulting heterodimeric Fc-part can befurther stabilized by artificial disulfide bridges.

An alternative approach is based on charged residues with ionicinteractions or steric complementarity. This includes altering thecharge polarity in the CH3 interface so that co-expression ofelectrostatically matched Fc domains support favorable attractiveinteractions and heterodimer formation while retaining the hydrophobiccore, whereas unfavorable repulsive charge interactions suppresshomodimerization. See Table 1. The amino acid numbering in Table 1follows the Kabat numbering scheme and can be applied to heavy chainamino acid sequences of the antibodies described herein.

In a further approach, the bispecific molecules of the presentapplication may be constructed with a non-immunoglobulin scaffoldcontaining leucine zipper (LZ) domains. A leucine zipper is a commonthree-dimensional structural motif in proteins, typically as part of aDNA-binding domain in various transcription factors. A single LZtypically contains 4-5 leucine residues at approximately 7-residueintervals, which forms an amphipathic alpha helix with a hydrophobicregion running along one side. In a particular embodiment, aheterodimeric protein scaffold comprises a LZ from the c-juntranscription factor associated with a LZ from the c-fos transcriptionfactor. Although c-jun is known to form jun-jun homodimers and c-fosdoes not form homodimers, the formation of jun-fos heterodimers isgreatly favored over jun-jun homodimers.

A leucine zipper domain may be incorporated in place of CH2-CH3sequences in the protein scaffold or it may be placed at the carboxyterminal end of the two heavy chains in the bispecific antitumorantagonist. In the case of the latter, a furin cleavage site may beintroduced between the carboxy terminal end of CH3 and the aminoterminal end of the leucine zipper. This can facilitate furin-mediatedcleavage of the leucine zipper following the heterodimerization stepwhen co-expressing the heavy and light chains of the bispecificantitumor antagonist in an appropriate mammalian cell expression system(see Wranik et al., J. Biol. Chem., 287(5):43331-43339, 2012).

TABLE 1 Type HC1 HC2 Knobs-into-holes Y349C, T366S, S354C, T366W L368A,Y407V Ionic, electrostatic S183E, E356K, S183K, K370E, E357K, D399KK409D, K439E Ionic, electrostatic K392D, K409D E356K, D399K HA-TFsubstitutions S364H, F405A Y349T, T394F HF-TA substitutions S364H, T394FY349T, F405A Leucine zipper human c-Jun leucine human c-fos leucineheterodimer zipper zipper

The amino acid numbering in Table 1 follows the Kabat numbering schemeand can be applied to heavy chain amino acid sequences of the antibodiesdescribed herein. The mutations described in Table 1 may be applied tothe sequence (published or otherwise) of any immunoglobulin IgG1 heavychain, as well as other immunoglobulin classes, and subclasses (orisotypes) therein.

When co-expressing heavy and light chains of bispecific antibodies, thelight chains of one binding specificity can also mispair with heavychains of a different binding specificity. Therefore, in certainembodiments, portions of the heavy chain, light chain or both may bemodified relative to the “wild-type” antibody chains from which they arederived to prevent or reduce mispairing of both heavy chain constantregions to one another, as well mispairing of light chain constantregions to their heavy chain counterparts.

The light chain mispairing problem can be addressed in several ways. Insome embodiments, sterically complementary mutations and/or disulfidebridges may be incorporated into the two VL/VH interfaces. In otherembodiments, mutations can be incorporated based on ionic orelectrostatic interactions. In some embodiments, light chain mispairingmay be prevented or reduced by employing a first arm with an S183Emutation in the CH1 domain of the heavy chain and an S176K mutation inthe CL domain of the light chain. A second arm may include an S183Kmutation in the in the CH1 domain of the heavy chain and an S176Emutation in the CL domain of the light chain. In other embodiments, a“CrossMab” approach is employed, where one arm in the bispecificantitumor antagonist (e.g., Fab) is left untouched, but in the other armcontaining the other binding specificity, one or more domains in thelight chain are swapped with one or more domains in the heavy chain atthe heavy chain: light chain interface.

Methods, immunoglobulin domain sequences, including specific mutationsfor preventing mispairing of heavy and light chains as disclosed aboveare further described in U.S. Patent Publication Nos. 2014/0243505 and2013/0022601.

Conjugates

In certain embodiments, the antitumor antagonists of the presentapplication are chemically conjugated to one or more peptides and/orsmall molecule drugs. The peptides or small molecule drug can be thesame or different. The peptides or small molecule drugs can be attached,for example to reduced SH groups and/or to carbohydrate side chains.Methods for making covalent or non-covalent conjugates of peptides orsmall molecule drugs with antibodies are known in the art and any suchknown method may be utilized.

In some embodiments the peptide or small molecule drug is attached tothe hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached usingheterobifunctional cross-linkers, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). General techniques for such conjugation arewell-known in the art. In some embodiments, the peptide or smallmolecule drug is conjugated via a carbohydrate moiety in the Fc regionof the antibody. The carbohydrate group can be used to increase theloading of the same agent that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different therapeutic ordiagnostic agent. Methods for conjugating peptide inhibitors or smallmolecule drugs to antibodies via antibody carbohydrate moieties arewell-known to those of skill in the art. For example, in one embodiment,the method involves reacting antibody component having an oxidizedcarbohydrate portion with a carrier polymer that has at least one freeamine function. This reaction results in an initial Schiff base (imine)linkage, which can be stabilized by reduction to a secondary amine toform the final conjugate. Exemplary methods for conjugating smallmolecule drugs and peptides to antibodies are described in U.S. PatentPublication No. 2014/0356385.

Preferably, the antitumor antagonists in the present disclosure retaincertain desirable characteristics and pharmacokinetic properties ofantibodies, including a desirable in vitro and in vivo stability (e.g.,lone half-life and shelf-life stability), efficient delivery intodesired target cells, increased affinity for binding partners, desirableantibody-dependent cell-mediated cytotoxicity and complement-dependentcytotoxicity, and reduced renal clearance or excretion. Accordingly,careful attention to size and need for particular constant regioneffector functions may be considered in the design of the antitumorantagonists.

The bispecific antitumor antagonists described herein may range in sizefrom 50 kD to 300 kD, from 50 kD to 250 kD, from 60 kD to 250 kD, from80 kDa to 250 kD, from 100 kD to 250 kD, from 125 kD to 250 kD, from 150kD to 250 kD, from 60 kD to 225 kD, from 75 kD to 225 kD, from 100 kD to225 kD, from 125 kD to 225 kD, from 150 kD to 225 kD, from 60 kD to 200kD, from 75 kD to 200 kD, from 100 kD to 125 kD to 200 kD, from 150 kDto 200 kD, from 60 kD to 150 kD, from 75 kD to 150 kD, from 100 kD to150 kD, from 60 kD to 125 kD, from 75 kD to 125 kD, from 75 kD to 100kD, or any range encompassed by any combination of whole numbers listedin the above cited ranges or any ranges specified by any combination ofwhole numbers between any of the above cited ranges.

Kits

The present application further provides a kit comprising a checkpointregulator antagonist or antitumor antagonist of the present application.In some embodiment, the kit comprises one or more bispecific immunecheckpoint regulators containing at least one TGFβ pathway inhibitorydomain. In some embodiments, the kit further contains additionalreagents, including secondary antibodies for detection, and additionalhuman antibodies described herein (e.g., a human antibody having acomplementary activity, which binds to a different epitope in the sameantigen, etc.). Kits typically include a label with instructionsindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

II. Methods of Using the Antitumor Antagonists

The antitumor antagonists of the present application have numerous invitro and in vivo utilities including, for example, enhancement ofimmune responses and treatment of cancers, infectious diseases orautoimmune diseases.

The antitumor antagonists of the present application may be administeredto cells in culture, in vitro or ex vivo, or to human subjects, e.g., invivo, to enhance immunity in a variety of diseases. Accordingly,provided herein are methods of modifying an immune response in a subjectcomprising administering to the subject antibody or antigen-bindingfragment thereof as described herein such that the immune response inthe subject is enhanced, stimulated or up-regulated. Preferred subjectsinclude human patients in whom enhancement of an immune response wouldbe desirable. The methods are particularly suitable for treating humanpatients having a disorder that can be treated by augmenting an immuneresponse (e.g., the T-cell mediated immune response). The methods areparticularly suitable for treatment of cancer or chronic infections invivo. For example, the antitumor antagonists may be administeredtogether with antigen of interest or the antigen may already be presentin the subject to be treated (e.g., a tumor-bearing or virus-bearingsubject) to enhance antigen-specific immunity. When antitumorantagonists are administered together with another agent, the two can beadministered separately or simultaneously.

In some embodiments, the checkpoint regulator antagonist in theabove-described method is an anti-PD-1 antibody, an anti-PD-L1 antibody,a VEGF antibody, a VEGFR antibody, or any fragment thereof incombination with a TGFβRII ECD, a VEGFR ECD, or both.

In any of the antibody embodiments described herein, the antibody ispreferably a human or humanized antibody.

Also encompassed within the scope of the present application are methodsfor detecting and/or measuring the presence of target molecule in asample comprising contacting the sample, and a control sample, with anantibody, antibody fragment, or bispecific antagonist of the presentapplication, which specifically binds to the target molecule underconditions that allow for formation of a complex between the antagonistand the target molecule. The formation of a complex is then detected,wherein a difference in complex formation between the sample compared tothe control sample is indicative the presence of target molecule in thesample.

Given the ability of the antitumor antagonist of the present applicationto block inhibition or co-inhibition of T cell responses, e.g.,antigen-specific T cell responses, provided herein are in vitro and invivo methods of using the antibodies described herein to stimulate,enhance or upregulate antigen-specific T cell responses, e.g.,anti-tumor T cell responses. In certain embodiments, CD3 stimulation isalso provided (e.g., by co-incubation with a cell expressing membraneCD3), which stimulation can be provided at the same time, before, orafter treatment with a checkpoint regulator antagonist. For example,provided herein are methods of enhancing antigen-specific T cellresponse comprising contacting said T cell with a checkpoint regulatorantagonist described herein, and optionally with CD3, such thatantigen-specific T cell response is enhanced, e.g., by removal of acheckpoint regulator mediated inhibitory effect. Any suitable indicatorof antigen-specific T cell response can be used to measure theantigen-specific T cell response. Non-limiting examples of such suitableindicators include increased T cell proliferation in the presence of theantibody and/or increase cytokine production in the presence of theantibody. In a preferred embodiment, interleukin-2 and/or interferon-γproduction by the antigen-specific T cell is enhanced.

Further encompassed in the present application is a method for enhancingan immune response (e.g., antigen-specific T cell response) in a subjectby administering a bispecific antitumor antagonist described herein tothe subject such that an immune response (e.g., antigen-specific T cellresponse) in the subject is enhanced. In a preferred embodiment, thesubject is a tumor-bearing subject and an immune response against thetumor is enhanced. A tumor may be a solid tumor or a liquid tumor, e.g.,a hematological malignancy. In certain embodiments, a tumor is animmunogenic tumor. In other embodiments, a tumor is non-immunogenic. Incertain embodiments, a tumor is PD-L1 positive. In other embodiments atumor is PD-L1 negative. A subject may also be a virus-bearing subjectin which an immune response against the virus is enhanced as aconsequence of administering the bispecific antitumor antagonist asdescribed herein.

In one embodiment, a method for inhibiting the growth of tumor cells ina subject comprises administering to the subject a bispecific antitumorantagonist described herein such that growth of the tumor is inhibitedin the subject. Also provided are methods of treating chronic viralinfection in a subject comprising administering to the subject abispecific antitumor antagonist as described herein such that thechronic viral infection is treated in the subject.

Also encompassed herein are methods for depleting T_(reg) cells from thetumor microenvironment of a subject with a tumor, e.g., cancerous tumor,comprising administering to the subject a therapeutically effectiveamount of a bispecific antitumor antagonist described herein thatcomprises an Fc that stimulates depletion of T_(reg) cells in the tumormicroenvironment. An Fc may, e.g., be an Fc with effector function orenhanced effector function, such as binding or having enhanced bindingto one or more activating Fc receptors.

In a preferred embodiment, T_(reg) depletion occurs without significantdepletion or inhibition of T_(eff) in the tumor microenvironment, andwithout significant depletion or inhibition of T_(eff) cells and T_(reg)cells outside of the tumor microenvironment. In certain embodiments, thesubject has higher levels of checkpoint regulator(s) on T_(reg) cellsthan on T_(eff) cells, e.g., in the tumor microenvironment. In certainembodiments, the bispecific antagonists may deplete T_(regs) in tumorsand/or T_(regs) in tumor infiltrating lymphocytes (TILs). For example,in a CT26 tumor model, an anti-mouse TIGIT antibody formatted as a mouseIgG2a (which exhibits effector function) was found to partially depleteboth Treg and CD8⁺ T cells, but did not deplete CD4⁺ T cells. Aneffectorless counterpart antibody or antagonist formatted as a mouseIgG1 D265A, did not deplete T cells.

In certain embodiments, the bispecific antitumor antagonist describedherein is given to a subject as an adjunctive therapy. Treatment ofcancer patient with a bispecific antitumor antagonist according to thepresent application may lead to a long-term durable response relative tothe current standard of care; long term survival of at least 1, 2, 3, 4,5, 10 or more years, recurrence free survival of at least 1, 2, 3, 4, 5,or 10 or more years. In certain embodiments, treatment of a cancerpatient with bispecific antitumor antagonist prevents recurrence ofcancer or delays recurrence of cancer by, e.g., 1, 2, 3, 4, 5, or 10 ormore years. Thus, treatment with these antagonists can be used as aprimary or secondary line of treatment.

In certain preferred embodiments, the subject has a cell proliferativedisease or cancer. Provided herein are methods for treating a subjecthaving cancer, comprising administering to the subject a bispecificantitumor antagonist described herein, such that the subject is treated,e.g., such that growth of cancerous tumors is inhibited or reducedand/or that the tumors regress. The bispecific antitumor antagonistsdescribed herein can be used alone to inhibit the growth of canceroustumors. Alternatively, any of these antitumor antagonists can be used inconjunction with another agent, e.g., other anti-cancer targets,immunogenic agents, standard cancer treatments, or other antibodies, asdescribed below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of a bispecificantitumor antagonist described herein. Preferably, the antibody containshuman or humanized immunoglobulin sequences.

Cancers whose growth may be inhibited using the antibodies of theinvention include cancers typically responsive to immunotherapy.Non-limiting examples of cancers for treatment include squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, squamousnon-small cell lung cancer (NSCLC), non NSCLC, glioma, gastrointestinalcancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer (e.g.,renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractoryprostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, andhead and neck cancer (or carcinoma), gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastaticmalignant melanoma, such as cutaneous or intraocular malignantmelanoma), bone cancer, skin cancer, uterine cancer, cancer of the analregion, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, cancer ofthe ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally-induced cancers including those induced by asbestos,virus-related cancers (e.g., human papilloma virus (HPV)-related tumor),and hematologic malignancies derived from either of the two major bloodcell lineages, i.e., the myeloid cell line (which produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cellline (which produces B, T, NK and plasma cells), such as all types ofleukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocyticand/or myelogenous leukemias, such as acute leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), andchronic myelogenous leukemia (CML), undifferentiated AML (MO),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NEIL), B-celllymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoidB-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma,anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-celllymphoma/leukemia, mantle cell lymphoma, angio immunoblastic T-celllymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primarymediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma,T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-celllymphoma, lymphoblastic lymphoma, post-transplantationlymphoproliferative disorder, true histiocytic lymphoma, primary centralnervous system lymphoma, primary effusion lymphoma, lymphoblasticlymphoma (LBL), hematopoietic tumors of lymphoid lineage, acutelymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides orSezary syndrome), and lymphoplasmacytoid lymphoma (LPL) withWaldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, lightchain myeloma, nonsecretory myeloma, smoldering myeloma (also calledindolent myeloma), solitary plasmocytoma, and multiple myelomas, chroniclymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, including fibrosarcomaand rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the centraland peripheral nervous, including astrocytoma, schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, hematopoietic tumors of lymphoid lineage, for exampleT-cell and B-cell tumors, including but not limited to T-cell disorderssuch as T-prolymphocytic leukemia (T-PLL), including of the small celland cerebriform cell type; large granular lymphocyte leukemia (LGL)preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head orneck, renal cancer, rectal cancer, cancer of the thyroid gland; acutemyeloid lymphoma, as well as any combinations of said cancers. Themethods described herein may also be used for treatment of metastaticcancers, refractory cancers (e.g., cancers refractory to previousimmunotherapy, e.g., with a blocking CTLA-4 or PD-1 antibody), andrecurrent cancers.

A bispecific antitumor antagonist of the present application can beadministered alone, in combination with another antitumor antagonist, orconcurrently with another antitumor antagonist. Alternatively, thebispecific antitumor antagonist can also be administered in combination,or concurrently with, an immunogenic agent, such as cancerous cells,tumor vaccines, purified tumor antigens (including recombinant proteins,peptides, and carbohydrate molecules), cells transfected with genesencoding immune stimulating cytokines, in a cancer vaccine strategy (Heet al. (2004) J. Immunol. 173:4919-28), or an oncolytic virus.

Many experimental strategies for vaccination against tumors have beendevised. In one of these strategies, a vaccine is prepared usingautologous or allogeneic tumor cells. Some of these cellular vaccineshave been shown to be most effective when the tumor cells are transducedto express GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90: 3539-43). Cancer vaccines have been shown toenhance effector T-cell infiltration into the tumors in preclinicalmodels. The major types of cancer vaccines include peptide vaccines,vector-based antigen specific vaccines, whole-cell vaccines, anddendritic cell vaccines. All vaccine-based therapies are designed todeliver either single or multiple antigenic epitopes or antigens fromthe whole cells to the patients and induce tumor-specific effector Tcells. Thus, a vaccine-based therapy may be the most efficient way toinduce T-cell infiltration into the tumor.

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host.

Inhibition of the checkpoint regulator pathway, TGF-β pathway, and/orangiogenesis pathways may be used in conjunction with a collection ofrecombinant proteins and/or peptides expressed in a tumor in order togenerate an immune response to these proteins. Such proteins may beviewed by the immune system as self-antigens and are therefore tolerantto them. The tumor antigen can include the protein telomerase, which isrequired for the synthesis of telomeres of chromosomes and which isexpressed in more than 85% of human cancers and in only a limited numberof somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumorantigens can also be “neo-antigens” expressed in cancer cells because ofsomatic mutations that alter protein sequence or create fusion proteinsbetween two unrelated sequences (i.e., bcr-abl in the Philadelphiachromosome), or idiotype from B cell tumors.

Non-limiting examples of tumor vaccines include sipuleucel-T(Provenge®), an FDA-approved tumor vaccine for metastatic prostatecancer; tumor cells transfected to express the cytokine granulocytemacrophage colony-stimulating factor (GM-CSF), such as the whole cellGM-CSF-secreting irradiated, allogeneic pancreatic cancer vaccine (GVAX;Johns Hopkins); a multi-peptide vaccine consisting of immunogenicpeptides derived from breast cancer antigens, neu, legumain, andp-catenin, which prolonged the vaccine-induced progression-free survivalof breast tumor-bearing mice when administered in combination withanti-PD-1 antibody (Karyampudi L. et al. (2014) Cancer Res74:2974-2985); peptides of melanoma antigens, such as peptides of gp100,MAGE antigens, Trp-2, MARTI and/or tyrosinase, or. Other tumor vaccinesinclude proteins from viruses implicated in human cancers such as humanpapilloma viruses (HPV)(e.g., Gardasil®, Gardasil 9@, and Cervarix®;hepatitis B virus (e.g., Engerix-B and Recombivax HB); hepatitis C virus(HCV), Kaposi's sarcoma associated herpes sarcoma virus (KSHV). Anotherform of tumor specific antigen that can be used in conjunction withTIGIT inhibition is purified heat shock proteins (HSP) isolated from thetumor tissue itself. These heat shock proteins contain fragments ofproteins from the tumor cells and these HSPs are highly efficient atdelivery to antigen presenting cells for eliciting tumor immunity.Talimogene laherparepvec (T-VEC, or Imlygic®) is an FDA-approvedoncolytic virus for the treatment of some patients with metastaticmelanoma that cannot be surgically removed.

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens, as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization may be effectively combined withcheckpoint regulator blocking to activate (unleash) more potentanti-tumor responses.

Inhibition of the checkpoint regulator pathway, TGF-β pathway, and/orangiogenesis pathways can also be combined with standard cancertreatments (e.g., surgery, radiation, and chemotherapy). In particular,checkpoint regulator inhibition can be effectively combined withchemotherapeutic regimes. In these instances, it may be possible toreduce the dose of chemotherapeutic reagent administered (Mokyr et al.(1998) Cancer Research 58: 5301-5304). An example of such a combinationis antitumor antagonist in combination with decarbazine for thetreatment of melanoma. Another example of such a combination is acheckpoint regulator antagonist or antitumor antagonist in combinationwith interleukin-2 (IL-2) for the treatment of melanoma. For example,the scientific rationale behind the combined use of checkpoint regulatorinhibition, TGF-β1/TGF-β1 RII inhibition, and/or angiogenesis inhibitionwith chemotherapy can promote cell death as a consequence of thecytotoxic action of most chemotherapeutic compounds, thereby resultingin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withcheckpoint regulator inhibition, TGF-β1/TGF-β1 RII inhibition, and/orangiogenesis inhibition through cell death are radiation, surgery, andhormone deprivation. Each of these protocols creates a source of tumorantigen in the host.

The bispecific antitumor antagonists described herein may also beconstructed to target Fcα or Fcγ receptor-expressing effector cells totumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Forexample, anti-Fc receptor/antitumor antigen (e.g., Her-2/neu) bispecificantibodies have been used to target macrophages to sites of tumor. Thistype of targeting may be adapted to the present embodiments to moreeffectively activate tumor specific responses. The T cell arm of theseresponses would be augmented by the inhibition of one or more checkpointregulator antagonists described herein. Alternatively, antigen may bedelivered directly to DCs by the use of bispecific antibodies that bindto tumor antigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofimmunosuppressive proteins expressed by the tumors. These include amongothers TGF-β, IL-10, and Fas ligand. Antibodies to each of theseentities can be used in combination with the antitumor antagonistsdescribed herein to counteract the effects of the immunosuppressiveagent and favor tumor immune responses by the host.

Other antibodies that activate host immune responsiveness can be used incombination with the antitumor antagonists described herein. Theseinclude molecules on the surface of dendritic cells that activate DCfunction and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity (Ridge et al. (1998)Nature 393: 474-478) and can be used in conjunction with the bispecificantagonists described herein. Activating antibodies to T cellcostimulatory molecules, such as OX-40 (Weinberg et al. (2000) Immunol164: 2160-2169), CD137/4-1BB (Melero et al. (1997) Nature Medicine 3:682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) mayalso provide for increased levels of T cell activation. In addition,inhibitors of other immune checkpoint regulators may also be used inconjunction with other antitumor antagonists described herein, asfurther described below.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, checkpoint regulator inhibition may beused to increase the effectiveness of the donor engrafted tumor specificT cells by reducing graft vs. tumor responses.

In certain embodiments, antitumor antagonist described herein may beadministered to a subject with an infectious disease, especially chronicinfections. In this case, similar to its application to cancer,antibody-mediated checkpoint regulator inhibition can be used alone, oras an adjuvant, in combination with vaccines, to enhance immuneresponsiveness to pathogens, toxins, and self-antigens. Exemplarypathogens for which this therapeutic approach can be applied include,but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes,Giardia, Malaria, Leishmania, Staphylococcus aureus, and Pseudomonasaeruginosa. Checkpoint regulator inhibition, TGF-β1/TGF-β1 RIIinhibition, and/or angiogenesis inhibition is particularly usefulagainst established infections by agents, such as HIV that present novelor altered antigens over the course of the infections. Administration ofbispecific antitumor antagonists can allow for recognition of theseantigens as foreign so as to provoke an appropriate T cell response.

Other pathogenic viruses causing infections treatable by the methodsdescribed herein include HIV, hepatitis (A, B, or C), herpesvirusinfections (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barrvirus), and infections caused by an adenovirus, influenza virus,flavivirus, echoviruses, rhinoviruses, coxsackie viruses, coronaviruses,respiratory syncytial viruses, mumps viruses, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus,arboviral encephalitis virus, or combination thereof.

Exemplary pathogenic bacteria or diseases caused therefrom which may betreatable by the methods described herein include Chlamydia, Rickettsia,Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococciand Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella,Diphtheria, Salmonella, Bacilli, Cholera, Leptospirosis tetanus,botulism, anthrax, plague, and Lyme disease.

Exemplary pathogenic fungi causing infections treatable by the methodsdescribed herein include Candida (e.g., albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g.,fumigatus, niger, etc.), Mucorales (e.g., mucor, absidia, rhizopus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Exemplary pathogenic parasites causing infections treatable by themethods described herein include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia Zambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, and Nippostrongylus brasiliensis.

In all of the above methods, checkpoint regulator inhibition,TGF-1/TGF-β1 RII inhibition, and/or angiogenesis inhibition can becombined with other forms of immunotherapy such as cytokine treatment(e.g., interferons, GM-CSF, G-CSF, IL-2), or bispecific antibody therapyusing two different binding specificities to provide enhancedpresentation of tumor antigens.

The bispecific antitumor antagonists described herein can be used toenhance antigen-specific immune responses by co-administration of one ormore of any of these antibodies with antigen of interest (e.g., avaccine). Accordingly, provided herein are methods of enhancing animmune response to antigen in a subject, comprising administering to thesubject: (i) the antigen; and (ii) the bispecific antitumor antagonist,such that an immune response to the antigen in the subject is enhanced.The antigen can be, for example, a tumor antigen, a viral antigen, abacterial antigen or antigen from a pathogen. Non-limiting examples ofsuch antigens include those discussed in the sections above, such as thetumor antigens (or tumor vaccines) discussed above, or antigens from theviruses, bacteria or other pathogens described above.

In certain embodiments, a peptide or fusion protein comprising theepitope to which the bispecific antitumor antagonist binds may be usedas a vaccine instead of, or in addition to, the antitumor antagonist(s).

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multi-specific antibodies or antagonists andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

Combination Therapies

In another aspect, the present application provides combinationtherapies for enhancing antigen-specific T cell response in a subject.In one embodiment, the method includes contacting a T cell withbispecific antitumor antagonist in combination with a second antibody,antibody fragment, antagonist or drug such that antigen-specific T cellresponse or apoptotic pathway is enhanced. For example, in someembodiments, the first bispecific antitumor antagonist specificallybinds to a first checkpoint regulator, such PD-1 or PD-L1, and a secondbispecific antitumor antagonist specifically binding to a differentcheckpoint regulator or to a different epitope. In some embodiments, thesecond antibody or antibody fragment comprises one or more differentHCVRs, LCVR, or CDR(s) from PD-1 and/or PD-L1.

In a related aspect, a method of reducing or depleting regulatory Tcells in a tumor of a subject in need thereof includes administering aneffective amount of antibody or antibody fragment in combination with asecond antibody, antibody fragment, antagonist or drug such that thenumber of regulatory T cells in the subject is reduced.

In some embodiments, the subject has a cell proliferative disease orcancer as described herein.

In other embodiments, the subject has a chronic viral infection,inflammatory disease or autoimmune disease as described herein.

The provision of two distinct signals to T-cells is a widely acceptedmodel for lymphocyte activation of resting T lymphocytes byantigen-presenting cells (APCs). This model further provides for thediscrimination of self from non-self and immune tolerance. The primarysignal, or antigen specific signal, is transduced through the T-cellreceptor (TCR) following recognition of foreign antigen peptidepresented in the context of the major histocompatibility-complex (MHC).The second or co-stimulatory signal is delivered to T-cells byco-stimulatory molecules expressed on antigen-presenting cells (APCs).This induces T-cells to promote clonal expansion, cytokine secretion,and effector function. In the absence of co-stimulation, T-cells canbecome refractory to antigen stimulation, which results in a tolerogenicresponse to either foreign or endogenous antigens.

In the two-signal model, T-cells receive both positive co-stimulatoryand negative co-inhibitory signals. The regulation of such positive andnegative signals is critical to maximize the host's protective immuneresponses, while maintaining immune tolerance and preventingautoimmunity. Negative signals seem necessary for induction of T-celltolerance, while positive signals promote T-cell activation. Bothco-stimulatory and co-inhibitory signals are provided to antigen-exposedT cells, and the interplay between co-stimulatory and co-inhibitorysignals is essential to controlling the magnitude of an immune response.Further, the signals provided to the T cells change as an infection orimmune provocation is cleared, worsens, or persists, and these changespowerfully affect the responding T cells and re-shape the immuneresponse.

The mechanism of co-stimulation is of therapeutic interest because themanipulation of co-stimulatory signals has shown to provide a means toeither enhance or terminate cell-based immune response. Recently, it hasbeen discovered that T cell dysfunction or anergy can occur concurrentlywith an induced and sustained expression of immune checkpointregulators, such as programmed death 1 polypeptide (PD-1) and itsligands, PD-L1 and PD-L2. PD-L1 is overexpressed in many cancers and isoften associated with poor prognosis (Thompson R H et al., Cancer Res2006, 66(7):3381). Further, the majority of tumor infiltrating Tlymphocytes predominantly express PD-1, in contrast to T lymphocytes innormal tissues and peripheral blood T lymphocytes indicating thatup-regulation of PD-1 on tumor-reactive T cells can contribute toimpaired antitumor immune responses (Blood 2009 114(8):1537). This maybe due to exploitation of PD-L1 signaling mediated by PD-L1 expressingtumor cells interacting with PD-1 expressing T cells to result inattenuation of T cell activation and evasion of immune surveillance.Inhibition of the PD-L1/PD-1 interaction provides a means to enhance Tcell immunity, including CD8+ T cell-mediated killing of cancer cellsand tumors. Similar enhancements to T cell immunity have been observedby inhibiting the binding of PD-L1 to the binding partner B7-1.Consequently, therapeutic targeting of PD-1 and other immune checkpointregulators are an area of intense interest.

Combining inhibition of TGF-β1 signaling, checkpoint regulatorsignaling, and/or angiogenesis signaling with other signaling pathwaysderegulated in tumor cells can provide a means for enhance treatmentefficacy. In recent years, a number of immune checkpoint regulators inthe form of receptors and their ligands have been identified. Oneimportant family of membrane-bound ligands that bind to co-stimulatoryor co-inhibitory receptors is the B7 family, which includes CTLA-4 andits ligands, B7-1 and B7-2; PD-1 and its ligands, PD-L1 (B7-H1) andPD-L2 (B7-DC); B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.Additional immune checkpoint antagonists include, but are not limited toTIM-3 and its ligand, Galectin-9; LAG-3 and its ligands, including liversinusoidal endothelial cell lectin (LSECtin) and Galectin-3; CD122 andits CD122R ligand; CD70, B7H3, B and T lymphocyte attenuator (BTLA), andVISTA (Le Mercier et al. (2015) Front. Immunol., (6), Article 418). Inaddition, a number of checkpoint regulator antagonists have beenidentified and tested in various clinical and pre-clinical models and/orapproved by the FDA (Kyi et al., FEBS Letters, 588:368-376 (2014). Theconcept of inhibitory receptor blockade, also known as immune checkpointblockade, has been validated by virtue of e.g., the FDA approval of thePD-1 inhibitors, nivolumab and pembrolizumab, as well as the anti-CTLA-4antibody, ipilimumab for metastatic melanoma.

An immune checkpoint antagonist modulates or interferes with theactivity of the immune checkpoint regulator so that, as a result of thebinding to the checkpoint regulator or its ligand, signaling through thecheckpoint regulator receptor is blocked or inhibited. By inhibitingthis signaling, immune-suppression can be reversed so that T cellimmunity against cancer cells can be re-established or enhanced. Incontrast, an immune checkpoint agonist (of e.g., a costimulatorymolecule) stimulates the activity of an immune checkpoint regulator sothat, as a result of the binding to the checkpoint regulator or itsligand, signaling through the checkpoint regulator receptor isstimulated. By stimulating this signaling, T cell immunity againstcancer cells can be re-established or enhanced.

Accordingly, in one embodiment, a method for stimulating an immuneresponse in a subject comprises administering to the subject abispecific antitumor antagonist described herein in combination withanother immune checkpoint regulator described herein above, such that animmune response is stimulated in the subject, for example to inhibittumor growth or to stimulate anti-viral response. In particular, thebispecific antitumor antagonist(s) can be administered as separateantagonists or as a multi-specific antagonist comprising bindingspecificities to multiple products.

In some embodiments, the bispecific antitumor antagonist of the presentapplication can be combined to stimulate an immune response with (i) anantagonist of the IgSF family protein, B7 family or TNF family thatinhibit T cell activation, or antagonist of a cytokine that inhibits Tcell activation (e.g., IL-6, IL-10, TGF-β, VEGF, or otherimmunosuppressive cytokines) and/or (ii) an agonist of a stimulatoryreceptors of the IgSF family, B7 family or TNF family or of cytokines tostimulate T cell activation, for stimulating an immune response. Inother embodiments, the subject is administered a bispecific antitumorantagonist in combination with anti-CTLA-4 antibody or CTLA-4antagonist. Exemplary anti-CTLA-4 antibodies for use in accordance withthe present application include ipilimumab, trevilizumab andtremelimumab.

In some embodiments, subjects with a cancer exhibiting high expressionof a ligand for an immune checkpoint regulator may be administered abispecific antagonist targeting a different checkpoint regulatorantagonist. By way of example, in one embodiment, a subject with acancer exhibiting high expression of PVR (CD155) and/or Nectin-2 (CD112)and/or low expression PD-L1 may be selected for monotherapy withanti-TIGIT or anti-LAG-3 antibodies or fragments thereof alone, or in acombination therapy with a PD-1 antagonist or other immune checkpointregulator antagonist. The bispecific antitumor antagonists of thepresent application may be co-administered with any additional agent(s),e.g., antibodies, antagonists, or drugs in amount(s) effective instimulating an immune response and/or apoptosis so as to furtherenhance, stimulate or upregulate an immune response and/or apoptosis ina subject.

In some embodiments, the bispecific antitumor antagonist is administeredsubsequent to treatment with a different antitumor antagonist. Forexample, in some embodiments, the bispecific antitumor antagonist of thepresent application may be administered only after treatment with amonospecific antitumor antagonist has failed, has led to incompletetherapeutic response, or there has been recurrence of the tumor orrelapse (such as “PD-1 failure”). In some embodiments, cancersexhibiting such failures may be screened for expression of e.g., PVRand/or Nectin-2 and only those having high level expression are treatedwith a bispecific antitumor antagonist of the present application.

In certain embodiments, the antitumor antagonist includes a dominantnegative protein domain of the immune checkpoint regulator. Inparticular embodiments, the dominant negative protein comprises anextracellular domain derived from a member selected from the groupconsisting of PD-L1, PD-L2, PD-1, B7-1, B7-2, B7H3, CTLA-4, LAG-3,TIM-3, TIGIT, BTLA, VISTA, CD70, and combinations thereof. In certainparticular embodiments, these extracellular domains are fused to animmunoglobulin constant region or Fc receptor in the presently describedantibodies. Such mutants can bind to the endogenous receptor so as toform a complex that is deficient in signaling. In certain embodiments,the extracellular domain is fused to an immunoglobulin constant regionor Fc fragment or to a monomer in the oligomeric protein complex.

In certain embodiments, a dominant negative PD-L1 antagonist comprisesthe extracellular domain of PD-L1, PD-L2, or PD-1. In anotherembodiment, a dominant-negative PD-1 antagonist is employed, which has amutation so that it is no longer able to bind PD-L1. An exemplarydominant negative protein is AMP-224 (co-developed by Glaxo Smith Klineand Amplimmune), a recombinant fusion protein comprising theextracellular domain of PD-L2 and the Fc region of human IgG.

Exemplary immune checkpoint regulator agonists include, but are notlimited to members of the tumor necrosis factor (TNF) receptorsuperfamily, such as CD27, CD40, OX40, GITR and 4-1BB (CD137) and theirligands, or members of the B7-CD28 superfamily, including CD28 and ICOS(CD278). Additional checkpoint regulator agonists include CD2, CDS,ICAM-1, LFA-1 (CD11a/CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C,SLAMF7, NKp80, CD160, B7-H3, CD83 ligand. Immune checkpoint agonists caninclude antibodies or soluble fusion protein agonists comprising one ormore costimulatory domains. Agonist antibodies include, but are notlimited to anti-CD40 mAbs, such as CP-870,893, lucatumumab, anddacetuzumab; anti-CD137 mAbs, such as BMS-663513 urelumab, andPF-05082566; anti-OX40 mAbs; anti-GITR mAbs, such as TRX518; anti-CD27mAbs, such as CDX-1127; and anti-ICOS mAbs.

Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. Nos. 6,111,090 and8,586,023; European Patent No.: 090505B1, PCT Publication Nos.: WO2010/003118 and WO 2011/090754. Anti-GITR antibodies are described in,e.g., in U.S. Pat. Nos. 7,025,962, 7,618,632, 7,812,135, 8,388,967, and8,591,886; European Patent Nos.: 1947183B1 and 1866339; PCT PublicationNos.: WO 2011/028683, WO 2013/039954, WO2005/007190, WO 2007/133822, WO2005/055808, WO 99/40196, WO 2001/03720, WO 99/20758, WO 2006/083289, WO2005/115451, WO 2011/051726. An exemplary anti-GITR antibody is TRX518.

Another family of membrane bound ligands that bind to co-stimulatory orco-inhibitory receptors is the TNF family of molecules that bind tocognate TNF receptor family members, which include CD40 and CD40L,OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137/4-1BB,TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR,LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1,Lymphotoxin α/TNF γ, TNFR2, TNFα, LTOR, Lymphotoxin α 1(32, FAS, FASL,RELT, DR6, TROY, NGFR (see, e.g., Tansey, M. G. et al. (2009) DrugDiscovery Today, 14(23-24):1082-1088).

Immune checkpoint agonists or costimulatory molecules include cellsurface molecules other than antigen receptors or their ligands that arerequired for an efficient immune response, and include, but are notlimited to MHC class I molecules, MHC class II molecules, TNF receptorproteins, immunoglobulin-like proteins, cytokine receptors, integrins,signaling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

In one aspect, T cell responses can be stimulated by a combination ofthe anti-PD-1 or anti-PD-L1 mAbs of the present invention and one ormore of (i) antagonist of a protein that inhibits T cell activation(e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1,PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1,CD113, GPR56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-1, CD96 andTIM-4, and (ii) an agonist of a protein that stimulates T cellactivation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40,ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents modulating one of the above proteins may be combinedwith the anti-PD-1 antibodies or anti-PD-L1 antibodies of the presentapplication for treating cancer, include e.g., YERVOY™/ipilimumab ortremelimumab (to CTLA-4), galiximab (to B7.1),OPDIVO™/nivolumab/BMS-936558 (to PD-1), pidilizumab/CT-01 (to PD-1),KEYTRUDA™/pembrolizumab/MK-3475 (to PD-1), AMP224 (to B7-DC/PD-L2),BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557(to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3), urelumab/BMS-663513 andPF-05082566 (to CD137/4-1BB), CDX-1127 (to CD27), MEDI-6383 andMEDI-6469 (to OX40), RG-7888 (to OX40L), Atacicept (to TACI), CP-870893(to CD40), lucatumumab (to CD40), dacetuzumab (to CD40), andmuromonab-CD3 (to CD3).

Other molecules that can be combined with the antitumor antagonistsdescribed herein for the treatment of cancer include antagonists ofinhibitory receptors on NK cells or agonists of activating receptors onNK cells. For example, antagonist anti-PD-1, and/or anti-PD-L1antibodies can be combined with antagonists of KIR (e.g., lirilumab),CSF-1R antagonists, such as RG7155.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofimmunosuppressive proteins expressed by the tumors. These include amongothers TGF-β, IL-10, and Fas ligand. Antibodies to each of theseentities can be used in combination with the antitumor antagonistsdescribed herein to counteract the effects of the immunosuppressiveagent and favor tumor immune responses by the host.

Other antibodies that activate host immune responsiveness can be used incombination with the antitumor antagonists described herein. Theseinclude molecules on the surface of dendritic cells that activate DCfunction and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity and can be used inconjunction with the antitumor antagonists described herein. Activatingantibodies to T cell costimulatory molecules, such as OX-40,CD137/4-1BB, and ICOS may also provide for increased levels of T cellactivation.

In certain embodiments, the antitumor antagonists described herein canbe co-administered with one or other more therapeutic agents, e.g.,anti-cancer agents, radiotoxic agents or an immunosuppressive agent.Such co-administration can solve problems due to development ofresistance to drugs, changes in the antigenicity of the tumor cells thatwould render them unreactive to the antagonist, and toxicities (byadministering lower doses of one or more agents).

The antitumor antagonists described herein can be linked to the agent(as an immuno-complex) or can be administered separate from the agent.In the latter case (separate administration), the antibody can beadministered before, after or concurrently with the agent or can beco-administered with other known therapies, e.g., anti-cancer therapy,e.g., radiation. The antitumor antagonists described herein may beco-administered with one or more anti-cancer agents so as to provide twoanti-cancer agents operating synergistically via different mechanisms toyield a cytotoxic effect in human cancer cells.

The antitumor antagonists described herein may be combined withanti-cancer agent, such an alkylating agent; anthracycline antibiotic;anti-metabolite; a detoxifying agent; an interferon; a polyclonal ormonoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histonedeacetylase inhibitor; a hormone; a mitotic inhibitor; aphosphatidylinositol-3-kinase (PI3K) inhibitor; an Akt inhibitor; amammalian target of rapamycin (mTOR) inhibitor; a proteasomal inhibitor;a poly(ADP-ribose) polymerase (PARP) inhibitor; a Ras/MAPK pathwayinhibitor; a centrosome declustering agent; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitor; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, anthracycline, a microtubule targeting drug, a topoisomerasepoison drug, an inhibitor of a molecular target or enzyme (e.g., akinase or a protein methyltransferase), a cytidine analogue orcombination thereof.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary anthracycline antibiotics include, but are not limited to,doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone(Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubidine);daunorubicin liposomal (DaunoXome); dactinomycin (Cosmegen); epirubicin(Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin(Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodinel31 tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Exemplary histone deacetylase inhibitors include, but are not limitedto, vorinostat (Zolinza), valproic acid, romidepsin, entinostatabexinostat, givinostat, and mocetinostat.

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron);abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary phosphatidyl-inositol-3 kinase (PI3K) inhibitors includewortmannin an irreversible inhibitor of PI3K, demethoxyviridin aderivative of wortmannin, LY294002, a reversible inhibitor of PI3K;BKM120 (Buparlisib); Idelalisib (a PI3K Delta inhibitor); duvelisib(IPI-145, an inhibitor of PI3K delta and gamma); alpelisib (BYL719), analpha-specific PI3K inhibitor; TGR 1202 (previously known as RP5264), anoral PI3K delta inhibitor; and copanlisib (BAY 80-6946), an inhibitorPI3Kα,δ isoforms predominantly.

Exemplary Akt inhibitors include, but are not limited to miltefosine,AZD5363, GDC-0068, MK2206, Perifosine, RX-0201, PBI-05204, GSK2141795,and SR13668.

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus;deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128,BEZ235, PI-103, Torin1, PP242, PP30, Ku-0063794, WAY-600, WYE-687,WYE-354, and CC-223.

Exemplary proteasomal inhibitors include, but are not limited to,bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib(CEP-18770), carfilzomib (PR-171), YU101, oprozomib (ONX-0912),marizomib (NPI-0052), and disufiram.

Exemplary PARP inhibitors include, but are not limited to, olaparib,iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016,MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338 andcombinations thereof.

Exemplary Ras/MAPK pathway inhibitors include, but are not limited to,trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026,RO4987655, RO5068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, andGDC-0973.

Exemplary centrosome declustering agents include, but are not limitedto, griseofulvin; noscapine, noscapine derivatives, such as brominatednoscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN),N-(3-brormobenzyl) noscapine, aminonoscapine and water-solublederivatives thereof, CW069; the phenanthridene-derived poly(ADP-ribose)polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furamide,N2-(2-thienylmethyl)-5-nitro-2-furamide, andN2-benzyl-5-nitro-2-furamide.

Exemplary multi-kinase inhibitors include, but are not limited to,regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080;Zd6474; PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol;seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412;bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244);SCIO-469; GW681323; CC-401; CEP-1707 or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 Mpotassium oxonate) or lovastatin.

In certain embodiments, the antitumor antagonists described herein areadministered at a subtherapeutic dose, another anti-immune checkpointregulator antibody or antagonist is administered at a subtherapeuticdose, the angiogenesis antagonist is administered at a subtherapeuticdose, or any antagonist in a combination thereof is each administered ata subtherapeutic dose.

In certain embodiments, inhibition of the TGFβ/TGFβ RII, checkpointregulator, and/or angiogenesis pathways may be combined with standardcancer treatments (e.g., surgery, radiation, and chemotherapy) inaccordance with conventional chemotherapeutic regimes. In theseinstances, it may be possible to reduce the dose of chemotherapeuticreagent administered. An example of such a combination is a checkpointregulator antagonist of the present application in combination withdecarbazine for the treatment of melanoma. Another example of such acombination is a checkpoint regulator antagonist of the presentapplication in combination with interleukin-2 (IL-2) for the treatmentof melanoma. It is believed that the combined use of checkpointregulator inhibition and chemotherapy can enhance apoptosis and increasetumor antigen presentation for cytotoxic immunity. Other synergisticcombination therapies include checkpoint regulator inhibition throughcell death when used in combination with radiation, surgery or hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host.

In certain embodiments, the checkpoint regulator antagonists describedherein can be used in multi-specific antagonists or in combination withbispecific antibodies targeting Fcα or Fcγ receptor-expressing effectorcells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti-tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tocancer cells or tumors. This targeting may more effectively activatetumor specific responses. The T cell arm of these responses would beaugmented by checkpoint regulator inhibition. Alternatively, antigen maybe delivered directly to DCs by the use of bispecific antibodies thatbind to tumor antigen and a dendritic cell specific cell surface marker.

III. Nucleic Acids and Host Cells for Expressing the AntitumorAntagonists

In another aspect, the present application provides nucleic acidsencoding the antitumor antagonists of the present application, includingthe heavy and light chains, as well as expression vectors comprisingsuch nucleic acids. In particular, the nucleic acids encode one or moreHCDRs, LCDRs, HCVRs and/or LCVRs corresponding to any of the antibodies,antagonists or fragments described herein.

Thus, in one aspect, the present application provides one or morenucleic acids encoding any of the antitumor antagonists, antibodies orantigen-binding portions thereof as described herein.

In another aspect, the present application provides one or moreexpression vectors comprising the one or more nucleic acids encoding anyof the antitumor antagonists, antibodies or antigen-binding portionsthereof as described herein.

In another aspect, the present application provides a host celltransformed with the one or more expression vectors comprising the oneor more nucleic acids encoding any of the antitumor antagonists,antibodies or antigen-binding portions thereof as described herein.

DNA(s) encoding antigen binding sites can be isolated and sequenced froma monoclonal antibody produced in hybridoma cells using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of themonoclonal antibodies). Alternatively, amino acid sequences fromimmunoglobulins of interest may be determined by direct proteinsequencing, and suitable encoding nucleotide sequences can be designedaccording to a universal codon table. In other cases, nucleotide andamino acid sequences of antigen binding sites or other immunoglobulinsequences, including constant regions, hinge regions and the like may beobtained from published sources well known in the art.

In one aspect, any of the binding antagonist fragments of the presentapplication may include the use of codon optimized synthetic DNAfragments corresponding to e.g., antibody variable domains etc. Forexample, the cDNA sequences encoding immunoglobulin VH, VL, HC, LC, CH1,CH2, CH3, and/or framework region can be codon optimized for expressionin various human, primate or mammalian cells, such as HEK or CHO cells.The invention also features a nucleic acid molecule that comprise one orboth nucleotide sequences that encode heavy and light chain variableregions, CDRs, hypervariable loops, framework regions of the anti-PD-L1antibody molecules, as described herein.

Expression vectors encoding a particular bispecific antitumor antagonistmay be used to synthesize the antitumor antagonists of the presentdisclosure in cultured cells in vitro or they may be directlyadministered to a patient to express the antitumor antagonist in vivo orex vivo. As used herein, an “expression vector” refers to a viral ornon-viral vector comprising a polynucleotide encoding one or morepolypeptide chains corresponding to the bispecific antitumor antagonistsof the present disclosure in a form suitable for expression from thepolynucleotide(s) in a host cell for antibody preparation purposes orfor direct administration as a therapeutic agent.

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, a DNA for a presequence or signal peptide isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a signalpeptide, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotideadaptors or linkers may be used in accordance with conventionalpractice.

Nucleic acid sequences for expressing the antitumor antagoniststypically include an amino terminal signal peptide sequence, which isremoved from the mature protein. Since the signal peptide sequences canaffect the levels of expression, the polynucleotides may encode any oneof a variety of different N-terminal signal peptide sequences. It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like.

The above described “regulatory sequences” refer to DNA sequencesnecessary for the expression of an operably linked coding sequence inone or more host organisms. The term “regulatory sequences” is intendedto include promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells or those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). Expression vectors generally contain sequences fortranscriptional termination, and may additionally contain one or moreelements positively affecting mRNA stability.

The expression vector contains one or more transcriptional regulatoryelements, including promoters and/or enhancers, for directing theexpression of antitumor antagonists. A promoter comprises a DNA sequencethat functions to initiate transcription from a relatively fixedlocation in regard to the transcription start site. A promoter containscore elements required for basic interaction of RNA polymerase andtranscription factors, and may operate in conjunction with otherupstream elements and response elements.

As used herein, the term “promoter” is to be taken in its broadestcontext and includes transcriptional regulatory elements (TREs) fromgenomic genes or chimeric TREs therefrom, including the TATA box orinitiator element for accurate transcription initiation, with or withoutadditional TREs (i.e., upstream activating sequences, transcriptionfactor binding sites, enhancers, and silencers) which regulateactivation or repression of genes operably linked thereto in response todevelopmental and/or external stimuli, and trans-acting regulatoryproteins or nucleic acids. A promoter may contain a genomic fragment orit may contain a chimera of one or more TREs combined together.

Preferred promoters are those capable of directing high-level expressionin a target cell of interest. The promoters may include constitutivepromoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α)) or thoseexhibiting preferential expression in a particular cell type ofinterest. Enhancers generally refer to DNA sequences that function awayfrom the transcription start site and can be either 5′ or 3′ to thetranscription unit. Furthermore, enhancers can be within an intron aswell as within the coding sequence. They are usually between 10 and 300bp in length, and they function in cis. Enhancers function to increaseand/or regulate transcription from nearby promoters. Preferred enhancersare those directing high-level expression in the antibody producingcell. Cell or tissue-specific transcriptional regulatory elements (TREs)can be incorporated into expression vectors to restrict expression todesired cell types. Pol III promoters (H1 or U6) are particularly usefulfor expressing shRNAs from which siRNAs are expressed. An expressionvector may be designed to facilitate expression of the antitumorantagonist in one or more cell types.

In certain embodiments, one or more expression vectors may be engineeredto express both the antitumor antagonist and one or more siRNA targetingthe Tie2 pathway, the VEGF pathway or an immune checkpoint regulator.

An siRNA is a double-stranded RNA that can be engineered to inducesequence-specific post-transcriptional gene silencing of mRNAs.Synthetically produced siRNAs structurally mimic the types of siRNAsnormally processed in cells by the enzyme Dicer. When expressed from anexpression vector, the expression vector is engineered to transcribe ashort double-stranded hairpin-like RNA (shRNA) that is processed into atargeted siRNA inside the cell. Synthetic siRNAs and shRNAs may bedesigned using well known algorithms and synthesized using aconventional DNA/RNA synthesizer.

To co-express the individual chains of the antitumor antagonist, asuitable splice donor and splice acceptor sequences may be incorporatedfor expressing both products. Alternatively, an internal ribosomebinding sequence (IRES) or a 2A peptide sequence, may be employed forexpressing multiple products from one promoter. An IRES provides astructure to which the ribosome can bind that does not need to be at the5′ end of the mRNA. It can therefore direct a ribosome to initiatetranslation at a second initiation codon within an mRNA, allowing morethan one polypeptide to be produced from a single mRNA. A 2A peptidecontains short sequences mediating co-translational self-cleavage of thepeptides upstream and downstream from the 2A site, allowing productionof two different proteins from a single transcript in equimolar amounts.CHYSEL is a non-limiting example of a 2A peptide, which causes atranslating eukaryotic ribosome to release the growing polypeptide chainthat it is synthesizing without dissociating from the mRNA. The ribosomecontinues translating, thereby producing a second polypeptide.

An expression vector may comprise a viral vector or a non-viral vector.A viral vectors may be derived from an adeno-associated virus (AAV),adenovirus, herpesvirus, vaccinia virus, poliovirus, poxvirus, aretrovirus (including a lentivirus, such as HIV-1 and HIV-2), Sindbisand other RNA viruses, alphavirus, astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,togaviruses and the like. A non-viral vector is simply a “naked”expression vector that is not packaged with virally derived components(e.g., capsids and/or envelopes).

In certain cases, these vectors may be engineered to target certaindiseases or cell populations by using the targeting characteristicsinherent to the virus vector or engineered into the virus vector.Specific cells may be “targeted” for delivery of polynucleotides, aswell as expression. Thus, the term “targeting”, in this case, may bebased on the use of endogenous or heterologous binding agents in theform of capsids, envelope proteins, antibodies for delivery to specificcells, the use of tissue-specific regulatory elements for restrictingexpression to specific subset(s) of cells, or both.

In some embodiments, expression of the antibody chains is under thecontrol of the regulatory element such as a tissue specific orubiquitous promoter. In some embodiments, a ubiquitous promoter such asa CMV promoter, CMV-chicken beta-actin hybrid (CAG) promoter, a tissuespecific or tumor-specific promoter to control the expression of aparticular antibody heavy or light chain or single-chain derivativetherefrom.

Non-viral expression vectors can be utilized for non-viral genetransfer, either by direct injection of naked DNA or by encapsulatingthe antitumor antagonist-encoding polynucleotides in liposomes,microparticles, microcapsules, virus-like particles, or erythrocyteghosts. Such compositions can be further linked by chemical conjugationto targeting domains to facilitate targeted delivery and/or entry ofnucleic acids into desired cells of interest. In addition, plasmidvectors may be incubated with synthetic gene transfer molecules such aspolymeric DNA-binding cations like polylysine, protamine, and albumin,and linked to cell targeting ligands such as asialoorosomucoid, insulin,galactose, lactose or transferrin.

Alternatively, naked DNA may be employed. Uptake efficiency of naked DNAmay be improved by compaction or by using biodegradable latex beads.Such delivery may be improved further by treating the beads to increasehydrophobicity and thereby facilitate disruption of the endosome andrelease of the DNA into the cytoplasm.

IV. Methods for Producing the Bispecific Antagonists

In another aspect, the present application provides host cellstransformed with the nucleic acids or expression vectors encoding thebispecific antitumor antagonists of the present application. The hostcells can be any eukaryotic or prokaryotic cell capable of expressingthe bispecific antitumor antagonists of the present application,including immunoglobulin heavy and light chains thereof.

In a further aspect, a method of producing antitumor antagonistcomprises culturing a host cell transformed with one or more nucleicacids or expression vectors encoding the bispecific antitumorantagonists of the present application under conditions that allow forproduction and purification of the antagonists, antibodies or fragmentsthereof expressed in suitable cells.

In a further aspect, the present application provides a method forproducing antibody comprising culturing a cell transiently or stablyexpressing one or more constructs encoding one or more polypeptidechains in the antibody; and purifying the antibody from the culturedcells. Any cell capable of producing a functional antibody may be used.In preferred embodiments, the antibody-expressing cell is of eukaryoticor mammalian origin, preferably a human cell. Cells from various tissuecell types may be used to express the antibodies. In other embodiments,the cell is a yeast cell, an insect cell or a bacterial cell.Preferably, the antibody-producing cell is stably transformed with avector expressing the antibody.

One or more expression vectors encoding the antibody heavy or lightchains can be introduced into a cell by any conventional method, such asby naked DNA technique, cationic lipid-mediated transfection,polymer-mediated transfection, peptide-mediated transfection,virus-mediated infection, physical or chemical agents or treatments,electroporation, etc. In addition, cells may be transfected with one ormore expression vectors for expressing the antibody along with aselectable marker facilitating selection of stably transformed clonesexpressing the antibody. The antibodies produced by such cells may becollected and/or purified according to techniques known in the art, suchas by centrifugation, chromatography, etc.

Examples of suitable selectable markers for mammalian cells includedihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycinanalog G418, hydromycin, and puromycin. When such selectable markers aresuccessfully transferred into a mammalian host cell, the transformedmammalian host cell can survive if placed under selective pressure.There are two widely used distinct categories of selective regimes. Thefirst category is based on a cell's metabolism and the use of a mutantcell line which lacks the ability to grow independent of a supplementedmedia. Two examples are CHO DHFR⁻ cells and mouse LTK⁻ cells. Thesecells lack the ability to grow without the addition of such nutrients asthymidine or hypoxanthine. Because these cells lack certain genesnecessary for a complete nucleotide synthesis pathway, they cannotsurvive unless the missing nucleotides are provided in a supplementedmedia. An alternative to supplementing the media is to introduce anintact DHFR or TK gene into cells lacking the respective genes, thusaltering their growth requirements. Individual cells which were nottransformed with the DHFR or TK gene will not be capable of survival innon-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, mycophenolic acid, orhygromycin. The three examples employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.Others include the neomycin analog G418 and puromycin.

Exemplary antibody-expressing cells include human Jurkat, humanembryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouseWEHI fibrosarcoma cells, as well as unicellular protozoan species, suchas Leishmania tarentolae. In addition, stably transformed, antibodyproducing cell lines may be produced using primary cells immortalizedwith c-myc or other immortalizing agents.

In one embodiment, the cell line comprises a stably transformedLeishmania cell line, such as Leishmania tarentolae. Leishmania areknown to provide a robust, fast-growing unicellular host for high levelexpression of eukaryotic proteins exhibiting mammalian-typeglycosylation patterns. A commercially available Leishmania eukaryoticexpression kit is available (Jena Bioscience GmbH, Jena, Germany).

In some embodiments, the cell line expresses at least 1 mg, at least 2mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, atleast 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or atleast 500 mg of the antibody/liter of culture.

The antibodies in the present application may be isolated from antibodyexpressing cells following culture and maintenance in any appropriateculture medium, such as RPMI, DMEM, and AIM V©. The antibodies can bepurified using conventional protein purification methodologies (e.g.,affinity purification, chromatography, etc.), including the use ofProtein-A or Protein-G immunoaffinity purification. In some embodiments,antibodies are engineered for secretion into culture supernatants forisolation therefrom.

V. Pharmaceutical Compositions and Methods of Treatment

Another aspect of the present application relates to pharmaceuticalcompositions and methods for treating a cell proliferative disorder,such as cancer, chronic infections, or immunologically compromiseddisease states. In one embodiment, the pharmaceutical compositioncomprises one or more antitumor antagonists of the present application.In some embodiments, the antitumor antagonist(s) comprise: one or moreTGF-β1 inhibitors or TGF-β1 RII inhibitors in combination with: (1) aPD-1 inhibitor or a PD-L1 inhibitor; or (2) or one or more angiogenesisinhibitors, such as VEGF inhibitors, VEGFR2 inhibitors, angiopoietin-1/2inhibitors, and Tie2R inhibitors. The antagonist(s) are formulatedtogether with a pharmaceutically acceptable carrier. Pharmaceuticalcomposition of the present application may include one or more differentantibodies, one or more multispecific antibodies, one or moreimmunoconjugates, or a combination thereof as described herein.

As described above, methods for using the pharmaceutical compositionsdescribed herein comprise administering to a subject in need thereof aneffective amount of the pharmaceutical composition according to thepresent disclosure.

Any suitable route or mode of administration can be employed forproviding the patient with a therapeutically or prophylacticallyeffective dose of the antibody or antagonist. Exemplary routes or modesof administration include parenteral (e.g., intravenous, intraarterial,intramuscular, subcutaneous, intratumoral), oral, topical (nasal,transdermal, intradermal or intraocular), mucosal (e.g., nasal,sublingual, buccal, rectal, vaginal), inhalation, intralymphatic,intraspinal, intracranial, intraperitoneal, intratracheal, intravesical,intrathecal, enteral, intrapulmonary, intralymphatic, intracavital,intraorbital, intracapsular and transurethral, as well as local deliveryby catheter or stent.

A pharmaceutical composition comprising antibody or antagonist inaccordance with the present disclosure may be formulated in anypharmaceutically acceptable carrier(s) or excipient(s). As used herein,the term “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Pharmaceutical compositions may comprisesuitable solid or gel phase carriers or excipients. Exemplary carriersor excipients include but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols. Exemplary pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of thetherapeutic agents.

The antitumor antagonist can be incorporated into a pharmaceuticalcomposition suitable for parenteral administration. Suitable buffersinclude but are not limited to, sodium succinate, sodium citrate, sodiumphosphate or potassium phosphate. Sodium chloride can be used to modifythe toxicity of the solution at a concentration of 0-300 mM (optimally150 mM for a liquid dosage form). Cryoprotectants can be included for alyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).Other suitable cryoprotectants include trehalose and lactose. Bulkingagents can be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants.

Therapeutic antitumor antagonist preparations can be lyophilized andstored as sterile powders, preferably under vacuum, and thenreconstituted in bacteriostatic water (containing, for example, benzylalcohol preservative) or in sterile water prior to injection.Pharmaceutical composition may be formulated for parenteraladministration by injection e.g., by bolus injection or continuousinfusion.

The therapeutic agents in the pharmaceutical compositions may beformulated in a “therapeutically effective amount” or a“prophylactically effective amount”. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the recombinant vector may varydepending on the condition to be treated, the severity and course of thecondition, the mode of administration, whether the antibody or agent isadministered for preventive or therapeutic purposes, the bioavailabilityof the particular agent(s), the ability of the antitumor antagonist toelicit a desired response in the individual, previous therapy, the age,weight and sex of the patient, the patient's clinical history andresponse to the antibody, the type of the antitumor antagonist used,discretion of the attending physician, etc. A therapeutically effectiveamount is also one in which any toxic or detrimental effect of therecombinant vector is outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result.

Preferably, the polypeptide domains in the antitumor antagonist arederived from the same host in which they are to be administered in orderto reduce inflammatory responses against the administered therapeuticagents.

The antitumor antagonist is suitably administered to the patent at onetime or over a series of treatments and may be administered to thepatient at any time from diagnosis onwards. The antitumor antagonist maybe administered as the sole treatment or in conjunction with other drugsor therapies useful in treating the condition in question.

As a general proposition, a therapeutically effective amount orprophylactically effective amount of the antitumor antagonist will beadministered in a range from about 1 ng/kg body weight/day to about 100mg/kg body weight/day whether by one or more administrations. In aparticular embodiment, each antitumor antagonist is administered in therange of from about 1 ng/kg body weight/day to about 10 mg/kg bodyweight/day, about 1 ng/kg body weight/day to about 1 mg/kg bodyweight/day, about 1 ng/kg body weight/day to about 100 μg/kg bodyweight/day, about 1 ng/kg body weight/day to about 10 μg/kg bodyweight/day, about 1 ng/kg body weight/day to about 1 μg/kg bodyweight/day, about 1 ng/kg body weight/day to about 100 ng/kg bodyweight/day, about 1 ng/kg body weight/day to about 10 ng/kg bodyweight/day, about 10 ng/kg body weight/day to about 100 mg/kg bodyweight/day, about 10 ng/kg body weight/day to about 10 mg/kg bodyweight/day, about 10 ng/kg body weight/day to about 1 mg/kg bodyweight/day, about 10 ng/kg body weight/day to about 100 μg/kg bodyweight/day, about 10 ng/kg body weight/day to about 10 μg/kg bodyweight/day, about 10 ng/kg body weight/day to about 1 μg/kg bodyweight/day, 10 ng/kg body weight/day to about 100 ng/kg body weight/day,about 100 ng/kg body weight/day to about 100 mg/kg body weight/day,about 100 ng/kg body weight/day to about 10 mg/kg body weight/day, about100 ng/kg body weight/day to about 1 mg/kg body weight/day, about 100ng/kg body weight/day to about 100 μg/kg body weight/day, about 100ng/kg body weight/day to about 10 μg/kg body weight/day, about 100 ng/kgbody weight/day to about 1 μg/kg body weight/day, about 1 μg/kg bodyweight/day to about 100 mg/kg body weight/day, about 1 μg/kg bodyweight/day to about 10 mg/kg body weight/day, about 1 μg/kg bodyweight/day to about 1 mg/kg body weight/day, about 1 μg/kg bodyweight/day to about 100 μg/kg body weight/day, about 1 μg/kg bodyweight/day to about 10 μg/kg body weight/day, about 10 μg/kg bodyweight/day to about 100 mg/kg body weight/day, about 10 μg/kg bodyweight/day to about 10 mg/kg body weight/day, about 10 μg/kg bodyweight/day to about 1 mg/kg body weight/day, about 10 μg/kg bodyweight/day to about 100 μg/kg body weight/day, about 100 μg/kg bodyweight/day to about 100 mg/kg body weight/day, about 100 μg/kg bodyweight/day to about 10 mg/kg body weight/day, about 100 μg/kg bodyweight/day to about 1 mg/kg body weight/day, about 1 mg/kg bodyweight/day to about 100 mg/kg body weight/day, about 1 mg/kg bodyweight/day to about 10 mg/kg body weight/day, about 10 mg/kg bodyweight/day to about 100 mg/kg body weight/day.

In other embodiments, the antitumor antagonist is administered at a doseof 500 g to 20 g every three days, or 25 mg/kg body weight every threedays.

In other embodiments, each antitumor antagonist is administered in therange of about 10 ng to about 100 ng per individual administration,about 10 ng to about 1 μg per individual administration, about 10 ng toabout 10 μg per individual administration, about 10 ng to about 100 μgper individual administration, about 10 ng to about 1 mg per individualadministration, about 10 ng to about 10 mg per individualadministration, about 10 ng to about 100 mg per individualadministration, about 10 ng to about 1000 mg per injection, about 10 ngto about 10,000 mg per individual administration, about 100 ng to about1 μg per individual administration, about 100 ng to about 10 μg perindividual administration, about 100 ng to about 100 μg per individualadministration, about 100 ng to about 1 mg per individualadministration, about 100 ng to about 10 mg per individualadministration, about 100 ng to about 100 mg per individualadministration, about 100 ng to about 1000 mg per injection, about 100ng to about 10,000 mg per individual administration, about 1 μg to about10 μg per individual administration, about 1 μg to about 100 μg perindividual administration, about 1 μg to about 1 mg per individualadministration, about 1 μg to about 10 mg per individual administration,about 1 μg to about 100 mg per individual administration, about 1 μg toabout 1000 mg per injection, about 1 μg to about 10,000 mg perindividual administration, about 10 μg to about 100 μg per individualadministration, about 10 μg to about 1 mg per individual administration,about 10 μg to about 10 mg per individual administration, about 10 μg toabout 100 mg per individual administration, about 10 μg to about 1000 mgper injection, about 10 μg to about 10,000 mg per individualadministration, about 100 μg to about 1 mg per individualadministration, about 100 μg to about 10 mg per individualadministration, about 100 μg to about 100 mg per individualadministration, about 100 μg to about 1000 mg per injection, about 100μg to about 10,000 mg per individual administration, about 1 mg to about10 mg per individual administration, about 1 mg to about 100 mg perindividual administration, about 1 mg to about 1000 mg per injection,about 1 mg to about 10,000 mg per individual administration, about 10 mgto about 100 mg per individual administration, about 10 mg to about 1000mg per injection, about 10 mg to about 10,000 mg per individualadministration, about 100 mg to about 1000 mg per injection, about 100mg to about 10,000 mg per individual administration and about 1000 mg toabout 10,000 mg per individual administration. The antitumor antagonistmay be administered daily, every 2, 3, 4, 5, 6 or 7 days, or every 1, 2,3 or 4 weeks.

In other particular embodiments, the amount of the antitumor antagonistmay be administered at a dose of about 0.0006 mg/day, 0.001 mg/day,0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day, 0.1mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day,30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day,2000 mg/day, 5000 mg/day or 10,000 mg/day. As expected, the dosage willbe dependent on the condition, size, age and condition of the patient.

In certain embodiments, the coding sequences for an antitumor antagonistare incorporated into a suitable expression vector (e.g., viral ornon-viral vector) for expressing an effective amount of the antitumorantagonist in patient with a cell proliferative disorder. In certainembodiments comprising administration of e.g., one or more recombinantAAV (rAAV) viruses, the pharmaceutical composition may comprise therAAVs in an amount comprising at least 10¹⁰, at least 10¹¹, at least10¹², at least 10¹³, or at least 10¹⁴ genome copies (GC) or recombinantviral particles per kg, or any range thereof. In certain embodiments,the pharmaceutical composition comprises an effective amount of therecombinant virus, such as rAAV, in an amount comprising at least 10¹⁰,at least 10¹¹, at least 10¹², at least 10¹³, at least 10¹⁴, at least10¹⁵ genome copies or recombinant viral particles genome copies persubject, or any range thereof.

Dosages can be tested in several art-accepted animal models suitable forany particular cell proliferative disorder.

Delivery methodologies may also include the use of polycationiccondensed DNA linked or unlinked to killed viruses, ligand linked DNA,liposomes, eukaryotic cell delivery vehicles cells, deposition ofphotopolymerized hydrogel materials, use of a handheld gene transferparticle gun, ionizing radiation, nucleic charge neutralization orfusion with cell membranes, particle mediated gene transfer and thelike.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

EXAMPLES Example 1: Generation of Monoclonal Antibodies

Monoclonal antibodies (mAbs) of the present application are generatedand screened using techniques well known in the art, see, e.g., Harlowand Lane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York. The antigen specific hybridoma mabs are cloned,sequenced and engineered using techniques well known in the art, seee.g., Lo. B. K. C Methods in Molecular Biology™. Volume 248 2004.Antibody Engineering.

Example 2: Design of Bispecific Antitumor Antagonists

FIGS. 5A and 5B show two bispecific antitumor antagonists, Bi-PB-1 (orBi-PLB-1) and Bi-PB-1 (or Bi-PLB-2), respectively. These antagonistscomprise a checkpoint regulator antibody backbone (anti-PD-1 oranti-PD-L1) along with a TGF-β RII extracellular domain (TGF-β-RII ECD):(i) fused to the carboxy-terminal end of the CH3 region in each of thetwo heavy chains (FIG. 5A) or (ii) inserted within the CH3 region of theFc loop (FIG. 5B).

In the embodiments depicted in FIGS. 5A and 5B, the bispecific antibodycan have an IgG1 or IgG4 backbone. Furthermore, any one or more of theantibody specificities may be substituted with any other checkpointregulator antagonist specificity and/or any tumor targeting antibodyspecificity, such as CD20, EGFR, etc.

FIG. 6 shows exemplary functional domain sequences corresponding to thebispecific antibodies in FIGS. 5A and 5B.

FIGS. 7A-7B show exemplary heavy chain (HC) and light chain (LC)sequences corresponding to the bispecific antibodies depicted in FIGS.5A and 5B.

Example 3: Design of Additional Bispecific Antagonists

FIGS. 8A-8C show the design of three different bispecific antagonists,Bi-AB-1, Bi-A1B-1 and Bi-ZB-1, respectively, each comprising acarboxy-terminal TGF-β1 RII extracellular domain (ECD) in a mutant IgG1(K447A) scaffold. Bi-AB-1 and Bi-A1B-1 both contain amino-terminalanti-VEGF variable regions (VH1, VL1) from Avastin/bevacizumab; Bi-A1B-1includes two amino acid substitutions in the VH region (E6Q, L11V).Bi-ZB-1 contains an amino terminal aflibercept domain upstream of anIgG1 Fc (K447A) region. In other embodiments, instead of a mutantIgG1(K447A) scaffold, the bispecific antagonists comprise a wild-typeIgG1 scaffold, a different mutant IgG1 scaffold, an IgG2 scaffold, amutant IgG2 scaffold, an IgG4 scaffold, or a mutant IgG4 scaffold.

FIGS. 9A and 9B show the various functional domain sequences present thebispecific antagonists depicted in FIGS. 8A-8C.

FIG. 10 shows the heavy chain (HC) and light chain (LC) amino acidsequences corresponding to the bispecific antagonists depicted in FIGS.8A-8C.

FIG. 11 summarizes the arrangement of functional domains in thebispecific antagonists depicted in FIGS. 8A-8C.

Example 4: Expression and Purification of the Antagonists in FIGS. 10-11

FIG. 12 is a Coomassie blue stained polyacrylamide gel showing improvedexpression levels of antagonists containing wt VEGF-A or mutant E6Q andL11V mutations (i.e., Bi-AB-1, Bi-A1B-1, respectively) when transientlytransfected into HEK293 cells as determined by non-reducingpolyacrylamide gel electrophoresis (PAGE). The titers depicted in FIG.12 were determined by quantifying the cell supernatant using a POROS Acolumn (Applied Biosystems). The results of this analysis showed thatthe expression level of Bi-A1B-1 was increased by 167% relative toBi-AB-1.

FIG. 13A depicts a size exclusion chromatography (SEC) profile showingrelative levels of high molecular weight (HMW), low molecular weight(LMW) species, and dimers resulting from protein A purified Bi-AB-1 andBi-A1B-1. FIG. 13B shows that the percentage of dimers (98.6% forBi-AB-1; 98.7% for Bi-A1B-1) greatly outnumber the HMW (1%) and LMW(0.4%) species purified as determined by SE-UPLC using Tosoh TSKgelUP-G3000SWXL columns. These results show that TGF-β1 RII extracellulardomain (ECD) fusions to Bi-AB-1 and Bi-A1B-1 did not result inappreciable levels of high molecular weight (HMW) or low molecularweight (LMW) species.

FIGS. 14A-164B depict Coomassie blue stained PAGE analyses of undernon-reducing and reducing conditions, respectively, from supernatantsobtained following transient transfection of Bi-ZB-1. The results ofthis analysis confirmed good transient expression levels of Bi-ZB-1 (220μg/ml) after 8 days of growth of cells in vitro.

FIG. 15 depicts a size exclusion chromatography (SEC) profile showingthat protein A purified Bi-ZB-1 has low levels of high molecular weight(HMW, 3.4%) and low molecular weight (LMW, 0.5%) species in comparisonto dimers (96.1%).

Example 5: Functional Characterization of the Antitumor Antagonists inFIGS. 8A-8C

To evaluate the ability of the anti-VEGF antagonist, Bi-A1B-1, toantagonize VEGF-mediated activation of VEGFR2 (i.e., the cognatereceptor), a cell-based luciferase assay was conducted. In thisexperiment, a recombinant HEK-293 cell line expressing human VEGFR2 anda firefly luciferase construct under the control of NFAT responseelements was stimulated with huVEGF165 in the presence of serialdilutions of anti-VEGF antagonist (Bi-A1B-1). The results of this assayare shown in FIG. 16. As expected, increasing levels of the anti-VEGFantagonist progressively neutralized the ability of VEGF165 to activateVEGFR2 and induce NFAT-mediated luciferase activity.

To evaluate the ability of the bispecific antagonist, Bi-A1B-1 tosimultaneously bind TGF-β1 and VEGF165, a sandwich ELISA assay wasperformed. In this experiment, huTGF-β1 was coated onto 96 well platesand blocked with 5% BSA, followed by addition and incubation of seriallydiluted samples of Bi-A1B-1, followed by addition of biotinylatedhuVEGF165. Bound molecules were detected by Streptavidin-HRP using a TMBsubstrate. The results of this analysis in FIG. 17 show that thebispecific antagonist, Bi-A1B-1 can simultaneously bind both TGF-β1 andVEGF165.

To evaluate the ability of the TGF-β1 RII ECD to antagonizeTGF-β1-mediated activation of the human TGF-β1 RII (i.e., the cognatereceptor), a cell-based luciferase assay was conducted. In thisexperiment, recombinant HEK-293 cells expressing human TGF-β1 RIIreceptor and a firefly luciferase construct under the control of a SMADresponse element was stimulated with huTGF-β1 in the presence of serialdilutions of antibodies fused to a TGF-β1 RII ECD (Bi-A1B-1, Bi-ZB-1).The results of this analysis are shown in FIG. 18. As expected,increasing levels of antibodies containing a TGF-β1 RII ECDprogressively neutralized the ability of TGF-β1 to activate the humanTGF-β1 RII and induce NFAT-mediated luciferase activity. In this case,Bi-A1B-1 and Bi-ZB-1 exhibited similar antagonism ability, as reflectedin an IC50 of 0.32 nM for Bi-A1B-1 and an IC50 of 0.31 for Bi-ZB-1.These IC50s were comparable to a control TGFBR2 fusion product(IC50=0.20 nM).

To evaluate the pharmacokinetic properties of Bi-A1B-1 in vivo,pharmacokinetic profiles were generated. Briefly, 10 mg/kg of eachantagonist was intravenously injected into the tail vein of 6-10 weekold female CD1 mice (n=2 mice per molecule). Serum was harvested at 3minutes, 3 hours, 1 day, 3 days, 7 days and 10 days post injection. Todetect the antibodies in the serum, 96 well ELISA plates were coatedwith 5 μg/ml goat anti-human IgG F(ab′)2 fragment and then blocked with5% milk in PBS. Serially diluted mouse serum in 5% milk and seriallydiluted purified protein molecule as standard were added to the plates.Following incubation with peroxidase conjugated mouse anti-human IgG andfurther washes, bispecific antagonist Bi-A1B-1 (FIG. 19) antibodies weredetected following incubation with TMB-ELISA substrate. The results ofthis analysis showed that the half-life (T2) of the bispecificantagonist Bi-A1B-1 is 7.4-16 days.

Example 6: Design of Bispecific PD-1/PD-L1 Antagonists with TGF-β1 ECDs

FIGS. 20A-20B depict two bispecific antitumor antagonists, Bi-PB-1.2(FIG. 20A) and Bi-PLB-1.2 (FIG. 20B), each comprising an antibodybackbone (IgG4 K447A or IgG1 K447A) with variable region domains fromanti-PD-1 and anti-PD-L1, respectively, and additionally including aTGF-β-RII ECD fused to the carboxy-terminal end of each heavy chain CH3region.

FIG. 21 shows functional domain sequences present in the bispecificantibodies in FIGS. 20A and 20B. FIG. 22 show the heavy chain (HC) andlight chain (LC) amino acid sequences corresponding to the bispecificantagonists depicted in FIGS. 20A and 20B. FIG. 23 summarizes thearrangement of functional domains in the bispecific antagonists depictedin FIGS. 20A and 20B.

Example 7: Expression and Purification of Bispecific Antagonists inFIGS. 20A-20B

FIG. 24A depicts a non-denaturing polyacrylamide gel (PAGE) analysisshowing expression of Bi-PB-1.2 and Bi-PLB-1.2 in a transient expressionsystem in comparison to 1 μg of purified a parental control antibody(2P17). FIG. 24B depicts size exclusion chromatography (SEC) profilesshowing that protein A purified Bi-PB-1.2, Bi-PLB-1.2 and anti-PDL1TGF-β-RII ECD fusion antibodies (Benchmark) from transiently transfectedHEK293 cells have low levels of high molecular weight (HMW) lowmolecular weight (LMW) species in comparison to dimerized molecules(“Dimers”), as shown in FIG. 24C. FIGS. 25A-25B show that the Dimer, HMWand LMW forms of Bi-PB-1.2 and Bi-PLB-1.2 exhibit good stability for atleast 4 weeks at 4° C.

Example 8: Functional Characterization of Bi-PB-1.2 and Bi-PLB-1.2Antagonists in FIGS. 20A-20B

1. Binding of Bi-PB-1.2 to PD-1 and TGF-β1 by Bio-Layer Interferometry

Bio-light interferometry was carried out using the Octet RED96 system(ForteBio Octet RED96 System) to characterize the binding kinetics ofantibodies against His tagged human PD-1 protein or His tagged TGF-β1.20 nM of antibody was loaded onto the anti-human IgG capture biosensors.Association of analyte (His tagged human PD-1 or TGF-β1 protein) wasobserved by placing the biosensors in wells containing 2 or 3 foldserial dilution of analytes (72 nM being the highest concentration) for5 mins. Dissociation was measured after transfer of the biosensors intokinetic buffer alone and monitoring of the interferometry signal for 10minutes. The observed on and off rates (Ka and Kd) were fit using a 1:1binding global fit model comprising at least 5 concentrations tested,and the equilibrium binding constant K_(D) was then calculated.

FIGS. 26A-26E show the results of PD-1 and TGF-β1 binding to Bi-PB-1.2(FIGS. 26A, 26B, respectively) and corresponding anti-PD-1 antibody andthe benchmark (M7824 from Merck KGA) (FIGS. 26C, 26D, respectively),along with their resultant binding affinity constants (FIG. 26E). Theresults show that the binding affinities of Bi-PB-1.2 for PD-1 and TGF-1are better than the anti-PD-1 benchmark and the M7824 benchmark.

2. Binding of Bi-PLB-1.2 to PD-L1 and TGF-β1 by Bio-Layer Interferometry

Binding of Bi-PLB-1.2 to PD-L1 and TGF-β1 by bio-layer interferometrywas carried out as described for Bi-PB-1.2 above. FIGS. 27A-27E show theresults of PD-1 and TGF-β1 binding to Bi-PB-1.2 (FIGS. 27A, 27B,respectively) and corresponding anti-PD-1 and anti-PDL1 TGF-β-RII ECDfusion antibody (TGF-1 Benchmark) (FIGS. 27C, 27D, respectively), alongwith their resultant binding affinity constants (FIG. 27E). The resultsshow that the binding affinity of Bi-PLB-1.2 for TGF-1 is better thanthe TGF-1 ECD benchmark, and the binding affinity of Bi-PLB-1.2 forPD-L1 is comparable to the anti-PD-L1 benchmark.

3. Simultaneous Binding to PD-1/PD-L1 by Bi-PB-1.2 and Bi-PLB-1.2

FIG. 28A shows an ELISA assay demonstrating simultaneous binding ofTGF-β1 and PD-1 by Bi-PB-1.2 in which huTGF-β1 coated 96 well plateswere incubated with serially diluted samples of Bi-PB-1.2, followed byHis-tagged huPD-1, whereby bound molecules were detected by anti-Histag-HRP using a TMB substrate. FIG. 28B shows an ELISA assaydemonstrating simultaneous binding of TGF-β1 and PD-L1 by Bi-PLB-1.2 inwhich huTGF-β1 coated 96 well plates were incubated with seriallydiluted samples of Bi-PLB-1.2, followed by His-tagged huPD-L1, wherebybound molecules were detected by anti-His tag-HRP using a TMB substrate.The EC50 values reflect the half maximal effective concentrationsproducing a response halfway between the baseline and maximum responsewith respect to binding human PD-1 or PD-L1.

4. Bi-PB-1.2 Potently Blocks Binding of Both PD-L1 and TGF-β1 to theirReceptors

In FIG. 29A, a blocking assay was carried out to calculate IC50 valuescorresponding to ability of Bi-PB-1.2 to block the binding of PD-1 toPD-L1 in comparison to an anti-PD-1 benchmark antibody. Briefly, 2 foldserial dilutions of Bi-PB-1.2 or the benchmark anti-PD-1 antibody(highest Ab concentration: 128 nM; Triplicates for each mAb) wereprepared. Human PD-1 transfected CHO-KI cells were washed with FACSbuffer (0.5% BSA 2 mM EDTA in PBS) and re-suspended at a concentrationof 10⁶ cells/ml. FITC labeled human PD-L1-Fc protein was added to thehuman PD-1 transfected CHO-KI cells at a final concentration of 7 μg/mland mixed well. Without incubation, 2,000 of these CHO-KI cells (withPD-L1 Protein) in 20 μl FACS buffer was immediately added to a 96-wellround bottom plate and 20 μl or 2 fold serial diluted antibodies wereimmediately added to the cells and incubated at 4° C. for 30 mins. Thecells were then washed and re-suspended in 30 μl 7AAD solution; 35 μl10% neutral buffered formalin solution was then added and incubated for15 min. before analysis using the iQue intellicyt system. As expected,the results of this analysis in FIG. 29A showed that increasing levelsof Bi-PB-1.2 progressively block the ability of PD-L1 to bind to itsreceptor. Further, the calculated IC50 of Bi-PB-1.2 for PD-L1 was foundto be comparable to the IC50 of the anti-PD-1 benchmark for PD-L1.

To evaluate the ability of the TGF-β1 RII ECD to antagonizeTGF-β1-mediated activation of the human TGF-β1 RII (i.e., the cognatereceptor), a cell-based luciferase assay was conducted. In thisexperiment, recombinant HEK-293 cells expressing human TGF-β1 RIIreceptor and a firefly luciferase construct under the control of a SMADresponse element was stimulated with huTGF-β1 in the presence of serialdilutions of Bi-PB-1.2 containing a TGF-β1 RII ECD. Bioactivity wasdetermined by a decrease in the amount of luciferase-mediatedluminescence. The results of this assay are shown in FIG. 29B. Asexpected, increasing levels of Bi-PB-1.2 and the benchmark anti-PD-L1TGF-1l RII ECD progressively neutralized the ability of TGF-β1 toactivate the human TGF-β1 RII and induce NFAT-mediated luciferaseactivity. Further, the calculated IC50 Bi-PB-1.2 for TGF-β1 was found tobe comparable to the IC50 of the anti-PD-L1 TGF-β1 RII ECD benchmark forTGF-β 1.

5. Bi-PB-1.2 Binds Both Human and Cynomolgus PD-1 with Similar Activity

To evaluate the ability of Bi-PB-1.2 to bind both human and cyno PD-1,serial dilutions of Bi-PB-1.2 were added to CHO-K1 cells (20,000cells/well) overexpressing human or cyno PD-1. The mixtures wereincubated at 4° C. for 20 min, washed 3 times, and stained with thesecondary antibody, PE labeled F(ab′)2-Goat anti-human IgG Fc (ThermoH10104) at 4° C. for 20 min. Cells were washed and resuspended in 7AADsolution and fixed in 10% neutral buffered formalin solution for 15minutes before analysis with the iQue Intellicyt system. FIGS. 30A-30Bshow binding of Bi-PB-1.2 to both human PD-1 (FIG. 30A) and cyno PD-1(FIG. 30B). As expected, the anti-human PD-1 benchmark antibody wassimilarly found to bind human PD-1 (FIG. 30A) and cynomolgus PD-1 (FIG.30B). Corresponding EC50 values reflecting the half maximal effectiveconcentrations (EC₅₀) producing a response halfway between the baselineand maximum response with respect to binding human PD-1 and cyno PD-1,respectively, were also determined. In this case, the EC50 values showsimilar binding properties of Bi-PB-1.2 as the benchmark antibody.

6. Bi-PLB-1.2 Potently Blocks Binding of Both PD-1 and TGF-β1 to theirReceptors

A blocking assay was carried out to calculate IC50 values correspondingto the binding between Bi-PLB-1.2 and PD-L1 in comparison to anti-PD-L1benchmark antibody, essentially as described in Example 8, section 4above. FIG. 31A shows that increasing concentrations of Bi-PLB-1.2 oranti-PD-L1 benchmark antibody progressively blocked binding of PD-1 toPD-L1. As reflected in the IC50 values obtained, the bispecific arecomparable to the anti-PD-L1 benchmark.

To calculate IC50 values corresponding to the binding between Bi-PLB-1.2and TGF-β1 in comparison to anti-TGF-β1 benchmark antibody, thecell-based assay described in Example 8, section 4 was carried out. FIG.31B shows the ability of serial dilutions of Bi-PLB-1.2 andanti-PDL1-TGF-β1 RII ECD benchmark to block the ability of TGF-β1 toactivate luciferase expression under the control of a SMAD responseelement. As reflected in the IC50 values obtained, the bispecificantibody exhibited similar binding characteristics as theanti-PDL1-TGF-β1 RII ECD benchmark.

7. Bi-PLB-1.2 Binds Both Human and Cynomolgus PD-1 with SimilarActivities

To evaluate the ability of Bi-PLB-1.2 to bind both human and cynomolgusPD-L1, serial dilutions of Bi-PLB-1.2 were added to CHO-K1 cells (20,000cells/well) overexpressing human or cyno PD-L1, essentially as describedin Example 9, section 5. FIGS. 32A-32B show that Bi-PLB-1.2 bound bothhuman PD-L1 (FIG. 32A) and cyno PD-L1 (FIG. 32B). The resulting EC50values reflect the half maximal effective concentrations (EC₅₀)producing a response halfway between the baseline and maximum responsewith respect to binding human PD-1 and cyno PD-1, respectively, werealso determined.

8. Bi-PB-1.2 Enhances T Cell Activation

Human T cell assays were performed to show the functionality of theBi-PB-1.2 antibodies. Briefly, normal healthy human PBMC collected fromDonors 1 and 2 were each activated with SEB (Toxin Technology, Cat#:BT202). To assay for IFN-γ production, 100,000 cells were stimulatedwith 0.5 ug/ml SEB in a 96 well plate. 25,000 tumor suppressing, SHP77cells were added to provide inhibitory signals. 66 nM of Bi-PB-1.2 mAbsor isotype control Ab was added. 5 days later, the supernatant wasexamined for IFN-γ and IL-2 by ELISA. FIGS. 33A-33B show increased IFN-γ(Donor 1, 40A; Donor 2, FIG. 33B) and IL-2 (Donor 1, 40C; Donor 2, FIG.33D) with Bi-PB-1.2 relative to the negative control treatments.

9. Bi-PLB-1.2 Enhances T Cell Activation and Overcomes T CellSuppression by Tumor Cells Better than the Parental Anti-PD-L1 Antibody.

FIGS. 34A-34D and show increased IFN-γ secretion from human PBMCs (Donor8, FIG. 34A; Donor 9, FIG. 34B) with Bi-PLB-1.2 relative to the negativecontrol treatments and the parental anti-PD-L1 antibody. FIGS. 34C-34Dshow increased IL-2 section from human PBMCs (Donor 8, FIG. 34C; Donor9, FIG. 34D) with Bi-PLB-1.2 relative to the negative control treatmentsand the parental anti-PD-L1 antibody. These results are consistent withthe notion that Bi-PLB-1.2 overcomes T cell suppression by tumor cellsbetter than the parental anti-PD-L1 antibody.

10. Improved Pharmacokinetic Profile in Mice for Bi-PB-1.2 andBi-PLB-1.2 Compared to a Benchmark Antibody

To evaluate the pharmacokinetic properties of Bi-PB-1.2, Bi-PLB-1.2 anda benchmark antibody in vivo, pharmacokinetic profiles were generated.Briefly, 10 mg/kg of each antagonist was intravenously injected into thetail vein of 6-10 week old female CD1 mice (n=2 mice per molecule).Serum was harvested at 3 minutes, 3 hours, 1 day, 3 days, 7 days and 10days post injection. To detect the antibodies in the serum, 96 wellELISA plates were coated with 5 μg/ml goat anti-human IgG F(ab′)2fragment and then blocked with 5% milk in PBS. Serially diluted mouseserum in 5% milk and serially diluted purified protein molecule asstandard were added to the plates.

Following incubation with peroxidase conjugated mouse anti-human IgG andfurther washes, Bi-PB-1.2 (FIG. 35A), Bi-PLB-1.2 (FIG. 35C), and theanti-PDL1-TGF-β1 RII ECD benchmark (M8724) (FIG. 35E) were detectedfollowing incubation with TMB-ELISA substrate. The integrity andclipping of the injected antibodies Bi-PB-1.2 (FIG. 35B), Bi-PLB-1.2(FIG. 35D), and the benchmark (FIG. 35F) in the mouse serum were alsoanalyzed by anti-human Fc Western blot. The results of this analysisshow improved pharmacokinetic profiles in mice for Bi-PB-1.2 andBi-PLB-1.2 compared to the benchmark antibody.

Example 9: Investigation of Proteolytic Cleavage in TGFβ1 RII ECD Domain

The inventors of the present application have unexpectedly discoveredthat antagonists bearing a TGFβ1 RII ECD domain exhibit unacceptablelevels of proteolytic degradation or clipping over time upon storagewhen they are produced by stably transfected CHO cells. This isillustrated in FIG. 36, which shows that the percentage of low molecularweight (LMW) species of Bi-PB-1.2 (PD1/TGFβ1 RII ECD; see FIG. 20A)increase over time stored at 4° C., while the percentage of dimerspecies decreases over time as determined by SE-UPLC using Tosoh TSKgelUP-G3000SWXL columns.

Because the LMW forms had not been seen when the PD1/PDL1-TGFB RII ECDmolecules were produced by transiently transfected HEK293 cells, SECanalyses were performed to compare stable CHO produced benchmark andBi-PB-1.2 from 2 different CHO transfection pool to bi_PB1.2 that wasproduced from HEK293 cells (FIG. 37). As shown in FIG. 37, the SECprofiles illustrate a shoulder near the main peak, along with smallersecondary peaks for the molecules that were produced by CHO cells, butnot by HEK293 cells consistent with increased proteolytic cleavage (orclipping) of CHO produced molecules.

To further examine the specific nature of the cleavage(s), the Bi-PB-1.2antagonist was expressed in CHO-K1 cells and subjected tocation-exchange chromatography (CEX) in order to generate fractions forfurther analysis by liquid chromatography-mass spectrometry (LC-MS) toidentify site(s) associated with clipping (FIG. 38). The same analysiswas extended to Bi-A1B-1 (data not shown). For the LC-MS analysis,samples were enzymatically deglycosylated using PNGase F. The reducedintact proteins were then separated by reversed phase ultra-highperformance liquid chromatography (RP-UPLC) using TSKgel Phenyl-5PW.Mobile phase A (MPA) and mobile phase B (MPB) were 0.05% formic acid inwater, and 0.05% formic acid in acetonitrile, accordingly. The columneffluent was directed through an in-line UV detector and then into theESI source of an ESI-TOF (Time-of-flight) mass spectrometer. Data wasdeconvoluted using Waters Masslynx software. For sequence accuracyconfirmation, the experimental masses of the reduced deglycosylatedmolecules were compared with theoretical masses calculated from theprimary amino acid sequence.

The results from LC-MS (data not shown) identified several proteolyticcleavage fragments (FIG. 39). More particularly, the results of theseanalyses showed that Bi-PB-1.2 and Bi-AB-1 have similar cleavage sitesin their respective heavy chains as further shown in FIGS. 40A and 40B,respectively.

Example 10: Characterization of TGFβ1 RII ECD Mutants to Reduce Clipping

In order to develop TGFβ1 RII ECD-containing variant moleculesexhibiting reduced clipping, various ECD mutants were designed as shownin FIG. 41A-41C. The mutants were designed to target the proposedclipping sites. FIGS. 42A-42H, 43, and 44A-44D show the heavy chainsequences of Bi-PB-1.2, Bi-PLB-1.2, and Bi-A1B-1 variants, respectively,containing the different ECD mutants described in FIGS. 41A-41C, whichwere generated and expressed. FIG. 45 highlights specific amino aciddeletion mutants without additional substitutions (variants Δ7, Δ12,Δ13) and with additional substitutions (variants B, C, I-B1), as well assubstitution mutants in the full length molecule (variants D-H)targeting the proposed clipping sites.

The resulting molecules were transiently expressed in CHO cells andprotein A affinity purified. In FIGS. 46A-46C, reduced PAGE analysis ofthe expressed products in FIGS. 42A-42H indicates a reduction ofclipping in the variant molecules. Consistent with this result, SECprofiles of the expressed products shown in FIGS. 47A and 47B are notaccompanied by “shoulders” and secondary peaks seen in Bi-PB-1.2 or asobserved in FIG. 37. In FIG. 48, peaks in the SEC profiles arequantified and show that the variants exhibit low percentages of highmolecular forms compared to the parental molecule.

Example 11: Functional Activity of TGFβ1 RII ECD Mutants

To confirm that that reduced clipping in the variants is not at theexpense functional activity, a cell-based assay was conducted toevaluate the functional activity of the ECD variants (FIG. 49). Briefly,serial dilutions of Bi-PB-1.2 and the various TGFβ1 RII ECD variantsthat were produced in HEK293 cells were made and tested (along with anegative control antibody) to see whether the antagonists block theability of TGF-β1 to activate luciferase expression under the control ofa SMAD response element as described above. Bioactivity was determinedby a decrease in the amount of luciferase-mediated luminescence. TheIC50 results of this analysis are summarized in FIG. 50, and show thatall of the variants retain bioactivities similar to the parentalBi-PB-1.2 and Bi-A1B-1 molecules, except Bi-A1B-1W, Bi-PB-1.2Δ15 andBi-PB-1.2Δ20.

Example 12: Pharmacokinetics of Bi-PB-1C and Bi-PB-1D

To evaluate the pharmacokinetic properties of Bi-PB-1.2C and Bi-PB-1.2Drelative to Bi-PB-1.2 (wt) in vivo, pharmacokinetic profiles weregenerated. Briefly, 10 mg/kg of each antagonist was intravenouslyinjected into the tail vein of 6-10 week old female CD1 mice (n=2 miceper molecule). Serum was harvested at 3 minutes, 3 hours, 1 day, 3 days,7 days and 10 days post injection. To detect the antibodies in theserum, 96 well ELISA plates were coated with 5 μg/ml goat anti-human IgGF(ab′)2 fragment (Sigma, #SAB3701274) and then blocked with 5% milk inPBS. Serially diluted mouse serum in 5% milk and serially dilutedpurified protein molecule as standard were added to the plates.Following incubation with Peroxidase AffiniPure Mouse Anti-Human IgG,Fcγ Fragment Specific (Jackson ImmunoResearch, #209-035-098) and furtherwashes, Bi-PB-1.2 (FIG. 51A), Bi-PB-1.2C (FIG. 51B), and Bi-PB-1.2D(FIG. 51C) antagonists were detected following incubation with TMB-ELISASubstrate Solution (Thermo-Fisher, #34029), and quantified by OD650signal with a Perkin Elmer multimode plate reader. The results of thisanalysis show pharmacokinetic profiles in mice for Bi-PB-1.2C andBi-PB-1.2D are comparable to Bi-PB-1.2, exhibiting an in vivo half-life(T_(1/2)) of about 5-8 days.

Example 13: Production from Stable CHO Cell Pools

The C variant molecules corresponding to parental Bi-PB-1.2, Bi-PLB-1,and Bi-A1B-1 molecules and the U variants of Bi-PB-1.2 and Bi-A1B-1molecules were produced from CHO-KI stable cell pools and their degreeof clipping was compared with their wild-type counterparts and/or withthe C variant counterparts. The CHO-K1 stable cell pools were grownunder fed-batch conditions for 14 days and purified by protein Achromatography. Clipping was assessed by reducing PAGE analysis of thepurified mutant and variant proteins. The results of this analysisshowed significant reduction of clipping with the C and U variants ascompared to the parental wild type molecules (FIGS. 52A-52B).

The above description is for the purpose of teaching a person ofordinary skill in the art how to practice the present invention and isnot intended to detail all those obvious modifications and variationswhich will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1.-20. (canceled)
 21. A protein comprising: a first targeting domaincomprising a mutated TGF-β1 RII extracellular domain (mutated ECD),wherein the mutated ECD comprises SEQ ID NO:89 with one or moresubstitutions and/or deletions that reduce proteolytic degradation orclipping; and an immunoglobulin scaffold having an amino terminus and acarboxy terminus, and wherein the mutated ECD comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 114-123 and251-270.
 22. The protein of claim 21, wherein the first targeting domainis linked to the immunoglobulin scaffold via a peptide linker.
 23. Theprotein of claim 21, further comprising a second targeting domain thatcomprises an immune checkpoint regulator antagonist, a VEGF pathwayantagonist, or a Tie2 tyrosine kinase receptor antagonist.
 24. Theprotein of claim 23, wherein the second targeting domain comprises animmune checkpoint regulator antagonist.
 25. The protein claim 24,wherein the second targeting domain specifically binds to PD-1.
 26. Theprotein claim 24, wherein the second targeting domain specifically bindsto PD-L1.
 27. The protein claim 24, wherein the second targeting domainspecifically binds to TIGIT.
 28. The protein claim 24, wherein thesecond targeting domain specifically binds to LAG-3.
 29. The protein ofclaim 23, wherein the second targeting domain comprises a VEGF pathwayantagonist.
 30. The protein of claim 29, wherein the VEGF pathwayantagonist specifically binds to VEGF-A.
 31. The protein of claim 29,wherein the VEGF pathway antagonist comprises VEGFR ECD.
 32. The proteinof claim 23, wherein the second targeting domain comprises a Tie2tyrosine kinase receptor antagonist.
 33. The protein of claim 22,wherein the second targeting domain comprises a SEQ ID NO: 157, 158,167, 168 or
 169. 34. The protein of claim 21, wherein the immunoglobulinscaffold is an IgG1, IgG2 or IgG4 scaffold.
 35. The protein of claim 34,wherein the immunoglobulin scaffold containing an N297A mutation, aK447A mutation, or both.
 36. The protein of claim 34, wherein theimmunoglobulin scaffold comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 100, 159-166 and 173-175.
 37. Apharmaceutical composition comprising: the protein of claim 1; and apharmaceutically acceptable carrier.
 38. An expression vector comprisinga nucleotide sequence encoding the protein of claim
 21. 39. A cell linethat expressed the protein of claim
 21. 40. A method of treating a cellproliferative disorder in a subject, comprising: administering to asubject in need thereof an effective amount of the protein of claim 21.